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Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation.
LDL receptor-deficient (LDLr−/−) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT−/−) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr−/− mice with TERT−/− bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at study endpoint without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT−/− bone marrow aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT−/− macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrate in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages.
TERT-deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr−/− mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.
Abdominal aortic aneurysms (AAA) represent a disease of an aging population with increasing incidence and considerable consequences on morbidity and mortality.1 Although remarkable progress has been made in recent years in understanding the pathogenetic mechanisms of AAA formation, there is currently no validated medical treatment option for this vascular disease.2–3 AAA are characterized as a chronic inflammatory and degenerative disease driven by wall proteolysis, inflammation, and oxidative stress.2 Particularly infiltrating macrophages in the aortic wall are a major source of matrix metalloproteinases (MMPs) and cytokines, leading to matrix degradation, further attraction of inflammatory cells, and induction of vascular smooth muscle cells apoptosis.4 For example, in the murine model of Ang II-induced AAA formation, we have previously demonstrated that medial accumulation of macrophages in areas of elastin degradation represents one of the earliest events in AAA formation.3
AAA are often only diagnosed at the time of rupture leading to a dramatic increase of morbidity and mortality.1 Therefore, early detection of AAA constitutes the mainstream of therapy, and the development of biomarkers may represent a possible solution for widespread screening.5 Although often confounded by lack of specificity, one biomarker that has been suggested to predict AAA formation is leukocyte telomere length.6–7 Telomeres, the DNA-protein complexes at the ends of eukaryotic chromosomes, are stabilized by the catalytic activity of the telomerase reverse transcriptase (TERT) to serve as protective capping.8 However, in the majority of adult somatic cells there is insufficient telomerase activity for telomere maintenance resulting in telomere attrition and impaired self-renewal.9 In contrast, stem cells and most cancer cells constitutively overexpress TERT manifesting high levels of telomerase activity, which confers an apparently indefinite lifespan.9 In addition to this well-established function of telomerase to elongate telomeres, recent evidence has identified a novel and unappreciated role for TERT in multiple developmental processes, including cell proliferation.10–12 Although still in its infancy, this novel function of TERT occurs independently of its catalytic activity to elongate telomeres and may involve a direct regulation of chromatin remodeling and gene transcription.10–12
While early studies suggested that TERT expression undergoes silencing in most organ systems, more recent work revealed that TERT is stringently regulated and induced in response to certain environmental cues.13 In vascular diseases, telomerase activity is induced 14–15 and reactivation of telomerase occurs in myeloid and lymphoid cells at critical stages of their activation, which has been suggested to enhance their function during inflammatory responses.16–17 In the present study, we investigated the causal role of TERT in inflammatory remodeling underlying AAA formation. Using bone marrow transplantation experiments, we demonstrate that TERT-deficiency attenuates AAA formation and decreases MMP-2 activity. These experiments establish a previously unrecognized causal role for TERT in the formation of AAA.
Bone marrow transplantation (BMT) studies were performed by repopulating lethally irradiated male LDL receptor-deficient mice (LDLr−/−, The Jackson Laboratory) with bone marrow-derived cells of male first generation TERT-deficient mice (TERT−/−, The Jackson Laboratory, B6.129S-Terttm1Yjc/J, stock number 005423) or their wild-type littermates (TERT+/+) as described.18 Briefly, mice were maintained on water containing antibiotics (sulfamethoxazole/trimethoprim) for 1 week prior to BMT until 4 weeks after BMT. Recipient mice were irradiated with a total of 900 Rads from a cesium source that was delivered in 2 doses within 3 to 4 hours. Bone marrow–derived cells of male TERT+/+ or TERT−/− mice were obtained from the tibias and femurs of donor mice and were injected into the tail vein of8-week-old irradiated male LDLr−/− recipient mice (1.2×107 cells per mouse, n = 10 with TERT+/+ and n = 7 with TERT−/−). After 4 weeks of recovery, a saturated fat-enriched diet was fed until the end of the study (Harlan Teklad TD.88137, Supplemental Table I). One week after initiation of this diet feeding, Ang II (1,000 ng/kg/min) was administered subcutaneously via Alzet osmotic minipumps (Model 2004) for 28 days. All studies were performed with the approval of the University of Kentucky Institutional Animal Care and Use Committee.
AAAs were quantified in vivo by measurement of maximal lumen diameters of suprarenal abdominal aortas using high-frequency ultrasound (Visualsonics) on anesthetized mice prior and at day 28 of Ang II infusion.19 AAAs were also quantified ex vivo by measurement of maximal width of suprarenal aortas dissected free from mice at 28 days and with extraneous tissues removed.20 En face atherosclerosis was quantified as lesion area on the intimal surface of aortic arches, as described previously.20
Serum cholesterol concentrations and lipoprotein cholesterol distributions were analyzed as described previously.21
Mouse abdominal aortas were fixed in formalin and embedded in OCT (Tissue-Tek, Miles Inc.). Serial 10-μm sections were cut surrounding the cross section of widest diameter. Aortic elastic laminae were stained with Verhoeff elastic staining. Macrophages were detected using adsorbed rabbit anti-mouse macrophage antisera (1:750 dilution; Accurate Chemicals).3
Differential leukocyte count was performed on whole blood at study endpoint using HEMAVET 950 hematology analyzer (Drew Scientific).
Mouse bone marrow macrophages (BMM) were isolated from the femurs of mice and differentiated using DMEM (Invitrogen) supplemented with 20% FBS and 30% L929 cell-conditioned medium for 12 days. For MMP-2 mRNA analysis and zymography, 8×105 cells/well were seeded on 6 well plates. Medium was changed to DMEM without supplements for 24 h and cells incubated with 1 μM Ang II for an additional 24 h. RAW 264.7 cells were obtained from American Type Culture Collection and maintained in DMEM supplemented with 10% FBS.
BMM were seeded at 1.5×105/well on a 24 well plate. Cells were kept in DMEM with 20% FBS for 24 h, stimulated with DMEM with 20% FBS and 30% L929 cell-conditioned medium and counted after 48 h using a hemocytometer. For BrdU-assays (Chemicon Inc.) cells were incubated with BrdU-reagent before stimulation with L929 cell-conditioned growth media. BrdU incorporation was analyzed after 24 h using a microplate reader according to the manufacturer’s instructions. All proliferation experiments were repeated at least 3 times and performed in triplicates.
BMM were treated with Colcemid, and metaphase spreads were prepared. Fluorescence in situ hybridization (FISH) was performed using the DAKO telomere FISH kit according to manufacturer’s instructions. For quantification of telomere length, DNA was isolated from BMM or whole blood using Qiagen DNeasy kit. Real-time PCR was performed using the method described by Cawthon.22 Samples were run in triplicates with 35 ng DNA per reaction using an iCycler and SYBR Green I system (Bio-Rad). Telomere repeat copy number data was normalized to 36B4 as single-copy gene. The primer sequences were: tel 1 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′, tel 2 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′; 36 B4 forward 5′-CAACCCAGCTCTGGAGAAAC-3′; reverse 5′-AAGCCTGGAAGAAGGAGGTC-3′.
RNA was isolated and reverse transcribed as described previously.23 Quantitative real-time PCR analysis of target gene expression was performed using an iCycler and SYBR Green I system (Bio-Rad). Each sample was analyzed in triplicate and normalized to mRNA expression of the house-keeping gene TFIIB.
The following primer sequences were used: MMP-2 forward: 5′-TGGGGGAGATTCTCACTTTG-3′; reverse: 5′-ATCACTGCGACCAGTGTCTG-3′ and TFIIB forward: 5′-CTCTCCCAAGAGTCACATGTCC-3′; reverse: 5′-CAATAACTCGGTCCCCTACAAC-3′.
Equal amounts of BMM supernatant or aortic protein were resolved in 7.5% SDS-PAGE containing 1 mg/ml gelatin (Sigma-Aldrich). Subsequently, SDS was removed from the gels by two washes (15 minutes) with 2.5% Triton X-100 (Sigma-Aldrich). Gels were incubated for 24 h (37°C) in zymography buffer (Tris-HCL 50 mM, Tris-Base 50 mM, CaClc2 5mM) and stained with Coomassie brilliant blue. Recombinant MMP-2 was used as positive controls (R&D Systems).
RAW 264.7 cells were cotransfected with a MMP-2 luciferase reporter construct 24 and a TERT expression vector 25 or a pcDNA3 vector as control using Lipofectamine 2000 (Invitrogen) as described.23 Transfection efficiency was normalized to renilla luciferase activities generated by cotransfection with pGL4.74[hRluc/TK] (Promega). Luciferase activity was assayed 24 h after transfection using a Dual Luciferase Reporter Assay System (Promega).
To compare two groups on a continuous response variable, we used a two-sample Student’s t-test or Mann-Whitney U test as appropriate. One-way ANOVA was used to compare multiple groups followed by Bonferroni’s post hoc analysis. All data are represented as means ± SEM.
To define whether telomerase in hematopoietic bone marrow cells affects AAA formation, LDLr−/− mice were lethally irradiated and reconstituted with bone marrow-derived cells isolated from TERT−/− mice or their wild-type littermates. As a model for AAA formation, mice were infused with Ang II for 28 days and suprarenal abdominal aortic diameters and areas were quantified in vivo by ultrasound and ex vivo using morphometric analysis.21 As depicted in Figure 1A–C, repopulation of LDLr−/− mice with TERT−/− bone marrow cells attenuated Ang II-induced AAA formation. The mean maximal suprarenal aortic diameters assessed by in vivo ultrasound were 1.17 mm and 1.00 mm in mice repopulated with TERT+/+ or TERT−/− bone marrow-derived cells, respectively (P < 0.01, Fig. 1A). Similarly, the mean aortic area was decreased in mice repopulated with TERT−/− bone marrow-derived cells compared to the wild-type littermates (0.78 mm2 vs. 1.10 mm2, respectively, P < 0.01, Fig. 1B). In contrast to mice expressing TERT in bone marrow-derived cells, Ang II-infusion did not increase the aortic diameter or area in recipients of TERT−/− bone marrow-derived cells compared to baseline (Supplemental Figure I). Ex vivo measurements of mean maximal aortic width confirmed an even greater difference between the donor genotypes (1.20 mm in mice repopulated with TERT+/+ bone marrow-derived cells vs. 0.95 mm in mice repopulated with TERT−/− bone marrow-derived cells, P < 0.001, Fig. 1C). Finally, TERT-deficiency in bone marrow-derived cells did not affect atherosclerosis formation in this model (Supplemental Figure II).
Ang II-induced aneurysm formation is associated with medial infiltration of macrophages and degradation of the aortic elastic laminae.3 To assess these histopathologic parameters in LDLr−/− mice repopulated with TERT−/− bone marrow-derived cells, we performed Verhoeff’s elastin staining and immunohistochemical analysis of macrophage infiltration. As depicted in Fig. 2A and B, mice repopulated with TERT+/+ bone marrow revealed aortic dilatation and frequent elastin breaks in the media. At these sites of elastin breaks, macrophages were abundantly identified. In contrast, mice reconstituted with TERT−/− bone marrow revealed decreased frequency of elastin breaks and accumulation of macrophages at these sites.
Compared to wild-type donors, repopulation of LDLr−/− mice with TERT−/− bone marrow cells revealed no significant effect on serum cholesterol concentrations or distributions (Supplemental Figure III and IV). Furthermore, no differences were observed in white blood cell counts 28 days after Ang II infusion (Table 1), confirming a sufficient repopulation of LDLr−/− mice with TERT−/− hematopoietic stem cells. Quantification of telomere lengths in circulating leukocytes in vivo revealed modest telomere shortening in mice repopulated with TERT−/− bone marrow, although this difference was not statistically significant (Fig. 3A). Similarly, in vitro analysis of telomere lengths by FISH and quantification using real-time PCR demonstrated unaltered telomere lengths in TERT−/− bone marrow-derived macrophages (Fig. 3B and C). To finally confirm a sufficient replicative capacity of TERT−/− bone marrow-derived macrophages in vitro, we next analyzed proliferation rates by means of cell counting and DNA synthesis. Consistent with the competent repopulation ability of TERT−/− hematopoietic cells in vivo, we observed no significant difference in proliferation rates between macrophages isolated from TERT-deficient mice and their wild-type littermates (Fig. 3D and E).
Since TERT-deficiency did not affect the proliferative capacity of hematopoietic stem cells or the number of circulating monocytes, the decreased AAA formation was likely due to altered leukocyte function rather than quantity. Among the mechanisms that promote AAA formation, MMP-2 function is critical due to its proteolytic activity to degrade extracellular matrix.26–28 Therefore, we next analyzed MMP-2 activity in aortic tissues isolated from LDLr−/− mice repopulated with TERT+/+ or TERT−/− bone marrow-derived cells and infused with Ang II for 28 days. As depicted in Fig. 4A and B, MMP-2 activity was significantly reduced in TERT−/− recipient mice. In vitro, incubation of wild-type bone marrow-derived macrophages with Ang II significantly increased MMP-2 mRNA abundance. In contrast, this inducible MMP-2 expression was decreased in TERT-deficient macrophages (Fig. 4C). Furthermore, the altered MMP-2 expression in TERT-deficient macrophages resulted in a corresponding decrease in MMP-2 activity as analyzed by zymography (Fig. 4D), demonstrating that TERT-deficiency in macrophages decreases MMP-2 activity. To finally address the mechanisms underlying the regulation of MMP-2 transcription by TERT, we performed transient transfection assays using a luciferase reporter construct driven by a 1686-kb MMP-2 promoter fragment. Consistent with the requirement of TERT for MMP-2 mRNA expression, overexpression of TERT significantly induced MMP-2 promoter activity (Fig. 4E).
AAA is a common and frequently lethal age-related vascular disease.2–3 Understanding the molecular mechanisms underlying this disease constitutes a critical step towards the development of a medical treatment option. 2 In the present study, we demonstrate that deletion of TERT in bone marrow cells attenuates AAA development in response to Ang II infusion of LDLr−/− mice. This decrease of AAA formation in mice lacking TERT expression occurred in the absence of a replicative defect of hematopoietic stem cells, but was associated with altered MMP-2 expression in macrophages. These data reveal an unanticipated causal role for TERT in the development of AAA.
Multiple human population studies have correlated decreased telomere length in peripheral blood leukocytes with an increased risk for atherosclerotic cardiovascular disease.29–30 Similarly, two recent studies have demonstrated decreased telomere length in leukocytes of patients with AAA.6–7 However, a causal relationship between loss of telomerase or telomere attrition and cardiovascular disease has never been established due to the lack of prospective studies.31 In fact, two recent large cohort studies found no correlation between telomere length and overall mortality, further challenging a causative link between telomere attrition and cardiovascular disease.32–33 Therefore, decreased telomere length in circulating leukocytes of patients with vascular disease may merely reflect an increased replicative history of hematopoietic stem cells necessary to mount the chronic inflammatory response in the arterial wall, particularly since leukocyte telomere shortening is observed in many other chronic inflammatory diseases 34–35 and occurs even during acute infections.36
Meanwhile animal models may provide initial insight into the causal role of telomerase and telomere attrition in vascular diseases. Using a TERT-deficient mouse model, we demonstrate in the present study that deletion of TERT in bone marrow cells reduces AAA formation. Mice deficient for TERT or the RNA-template TERC retain their telomeres in early generation offsprings, which has been attributed to the very long telomeres in mice.37–39 However, interbreeding of telomerase-deficient mice to yield successive generations results in progressive telomere attrition and ultimately degeneration of proliferative tissues in late generation mice.38–39 Therefore, hematopoietic stem cells from early generation telomerase-deficient mice display normal proliferation, while critically short telomeres in late generation mice impair progenitor cell proliferation and limit the self-renewal capacity.38–41 This observation has led to the conclusion that telomerase may be dispensable for stem cell proliferation if telomeres are not limiting.38, 40–41 Similarly, in our study using first generation TERT−/− mice, deficiency for the locus did not reduce telomere lengths or impair the proliferative capacity of bone marrow-derived macrophages in vitro. Moreover, hematopoietic stem cells from first generation TERT−/− mice effectively reconstituted irradiated mice in vivo, as evidenced by normal circulating leukocyte numbers and subpopulations. However, circulating leukocytes of mice repopulated with TERT−/− stem cells displayed mild telomere shortening, most likely as a result of the increased proliferation required to regenerate the stem cell compartment. Consistent with our studies, Allsopp et al. have demonstrated that stem cells from early generation TERT−/− mice exhibit normal telomere lengths and provide efficient hematopoietic recovery for two rounds of serial BMT, despite modest decreases in telomere lengths after the first transplantation. 41 Collectively, from these observations we would infer that the protection in AAA formation in mice reconstituted with TERT−/− bone marrow is likely the result of TERT-deficiency affecting leukocyte function rather than proliferation or quantity. Although the loss of telomere length in leukocytes of LDLr−/− mice reconstituted with TERT-deficient bone marrow was not statistically significant, we cannot exclude the possibility that leukocyte telomere attrition might have affected AAA formation in these mice. However, if this were the case, telomere attrition would rather protect against AAA formation as opposed to cause the disease. Our study was not designed to address the contribution of telomere length to AAA formation but rather investigated the role of TERT itself for the disease process. Considering this limitation, our data warrants further studies specifically investigating the contribution of progressive telomere attrition to AAA formation.
Alternatively and more likely, TERT may affect AAA formation through a mechanism that is distinct from its catalytic activity to extend telomeres. There is accumulating appreciation of a non-canonical telomere-independent function of TERT.42 Support for this novel function of TERT derives from recent experiments showing that TERT modulates the chromatin state,10, 12 interacts with DNA-modifying enzymes,12, 43 and activates gene expression,11–12, 44 effects that do not require catalytic activity and occur independently of telomere maintenance.11–12, 42 Considering this evidence, we reasoned that TERT-deficiency might modulate macrophage function and analyzed MMP expression. Degradation of extracellular matrix and elastolysis through the activities of MMPs constitute the best-described mechanisms underlying AAA formation. MMP-2 deficiency has been demonstrated to protect mice from AAA formation.28 Moreover, MMP-2 is the dominant metalloproteinase in the early stages of AAA formation causing the initial elastolysis,4, 45 a step driven primarily by macrophages in the Ang II-infusion model.3 In our studies, TERT-deficiency prevented Ang II-induced MMP-2 expression and resulted in reduced activity of secreted MMP-2. Conversely, TERT overexpression activated the MMP-2 promoter pointing to a transcriptional mechanism underlying the positive regulation of MMP-2 by TERT. Our observation that TERT is not only necessary for MMP-2 expression but also sufficient to activate MMP-2 transcription is consistent with a previous gain-of-function study demonstrating that overexpression of TERT increases MMP-2 levels in human fibroblasts.46
An intriguing question that arises from the observation that TERT induces MMP-2 promoter activity relates to the transcriptional mechanisms governing this regulation. The silencing of Ang II-induced MMP-2 expression in TERT-deficient macrophages and transactivation of the MMP-2 promoter occurred in cells with normal telomere length, supporting the concept of a non-canonical telomere length-independent mechanism. Several previously reported telomere-independent functions of TERT are conceivable to explain this observation. TERT has been shown to physically interact with NF-κB,47 which may positively regulate downstream MMP-2 expression. Alternatively, TERT may induce MMP-2 promoter activity through its recently described chromatin remodeling activity,10, 12, 48 particularly since an epigenetic regulation of the MMP-2 promoter has been well documented.49 Finally, TERT has recently been characterized as a transcriptional modulator of the Wnt/β-catenin signaling pathway.12 Interestingly, this study established that TERT interacts with the Brahma-related gene 1 (BRG1), the central catalytic subunit of numerous chromatin-modifying enzymatic complexes, resulting in the activation of Wnt-dependent promoters. Considering that MMP-2 constitutes a target gene of the Wnt-signaling pathway,50 TERT may induce MMP-2 transcription through a Wnt-dependent transcriptional activation. Our findings in the context of these intriguing studies provide justification for further defining the mechanism governing the transcriptional regulation of MMP-2 by TERT. In particular, further studies will be required to investigate telomere-dependent activities of TERT in vascular biology and functions that are mediated through non-canonical mechanisms independently of its reverse transcriptase function.
Acknowledgments - none
Sources of Funding - This work was supported by the American Recovery and Reinvestment Act of 2009 (NIH/NHLBI 1R01HL091924-01A1 to D.B.). F. Gizard and Y. Zhao were supported by Fellowship Grants from the American Heart Association (0725313B and 0815514D, respectively).
Disclosure - The authors have nothing to disclose that could be perceived as real or apparent conflict(s) of interest.