Telomerase mediates lymphocyte proliferation but not the atherosclerosis-suppressive potential of regulatory T-cells

Objective: Atherosclerosis is an age-related disease characterised by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence a potential causative role of telomerase in atherogenesis is critically debated. Approach and Results: In this study we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent, as oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase-knock-out mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase (TERT) deficiency had no effect on Treg numbers in vivo or suppressive function ex vivo. Adoptive transfer of TERT-/- Tregs into Rag2-/- ApoE-/- double knock out mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. Conclusions: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.This study was supported, in part, by British Heart Foundation Project Grants PG/15/85/31744 and PG/12/47/29681 (www.BHF.org.uk) as well as the Newcastle Healthcare Charity (www.newcastle-hospitals. org.uk/patient-guides/charity-matters-at-newcastle-hospitals_charitable- funds.aspx). N.M. Al Zhrany was funded by a stipend from the Government of Saudi Arabia

Telomerase mediates lymphocyte proliferation but not the atherosclerosis-suppressive potential of regulatory T-cells

Objective: Atherosclerosis is an age-related disease characterised by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence a potential causative role of telomerase in atherogenesis is critically debated. Approach and Results: In this study we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent, as oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase-knock-out mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase (TERT) deficiency had no effect on Treg numbers in vivo or suppressive function ex vivo. Adoptive transfer of TERT-/- Tregs into Rag2-/- ApoE-/- double knock out mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. Conclusions: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.This study was supported, in part, by British Heart Foundation Project Grants PG/15/85/31744 and PG/12/47/29681 (www.BHF.org.uk) as well as the Newcastle Healthcare Charity (www.newcastle-hospitals. org.uk/patient-guides/charity-matters-at-newcastle-hospitals_charitable- funds.aspx). N.M. Al Zhrany was funded by a stipend from the Government of Saudi Arabia

652The effect of varying irrigation flow rate during irrigated radiofrequency ablation on optimising lesion shape

Abstract Funding Acknowledgements Investigator Sponsored Research grant from Boston Scientific Introduction Irrigated catheters are the standard tool for radiofrequency (RF) ablation in the left atrium and ventricles. However, pathological studies of irrigated RF lesions show a "tear-drop" shape, with the widest diameter some depth below the endocardial surface and relative endocardial sparing. This requires overlap of lesions to achieve contiguity at the endocardial surface. There has been little investigation into the effect of altering irrigation rate on lesion shape and volume. Purpose To test the hypothesis that varying the irrigation flow rate would optimise lesion shape by minimising endocardial sparing while maintaining lesion depth. Methods In an ex vivo animal heart model, irrigated ablation lesions were performed in porcine ventricular tissue at 30W using an Intellatip MiFi OI catheter with 5 different irrigation protocols: A. fixed rate (30ml/min); B. continuous reduction (30 to 2ml/min over 30s); C. continuous increase (2 to 30ml/min over 30s); D. stepwise reduction (30ml/min (10s) to 16ml/min (10s) to 2ml/min (10s)); E. stepwise increase (2ml/min (10s) to 16ml/min (10s) to 30ml/min (10s). Contact force (10g) and ablation duration (30s) were constant. Steam pops during ablation were recorded. Tissue sections were stained with triphenyltetrazolium chloride (TTC) after ablation to allow accurate measurement of lesion boundaries. Surface diameter, lesion depth, maximum diameter, and depth at maximum diameter were measured using Vernier Calipers to calculate lesion volume. Pictures of lesions were analysed further by ImageJ software to measure the degree of endocardial sparing. The optimal protocol was further tested against fixed-rate irrigation at 20W – 40W. Results 10-20 lesions were performed for each irrigation protocol. Of the 4 experimental protocols, continuous reduction (protocol B) resulted in the most optimal lesion shape (Figure). With this protocol, endocardial sparing area was significantly reduced compared to fixed-rate irrigation (1.61 vs. 2.64mm2, P &amp;lt; 0.0001), with a trend towards an increase in surface diameter (9.25 vs. 8.46mm, P = 0.08). There was no significant difference in lesion depth (5.35 vs. 4.77mm), lesion volume (374 vs. 332mm3) or maximum diameter (10.3 vs. 10.8mm). Steam pop occurred in 1 of 20 (5%) lesions in each of protocols A and B. Significantly reduced endocardial sparing with preserved volume/depth was consistent for continuous reduction when compared to fixed-rate irrigation across power settings of 20W (17ml/min), 30 W (17ml/min or 30ml/min) and 40W (30ml/min) (P &amp;lt; 0.0001). Conclusions Continuous reduction in irrigation flow rate from 30 to 2ml/min during irrigated RF ablation results in reduced endocardial sparing with preserved lesion depth and volume when compared to fixed-rate irrigation across power settings of 20 – 40W. This may allow for greater lesion spacing while maintaining endocardial contiguity and merits further investigation to improve irrigated RF ablation efficiency. Abstract Figure </jats:sec