Highly Active Anti-Retroviral drugs increase poly (ADP-ribose) polymerase activation in cardiovascular cells resulting in cellular dysfunction

Peter Keltie1, Katherine Maple1, Mary El Assal2, Jon Mabley2
1Brighton & Sussex Medical School, Brighton, East Sussex, United Kingdom, 2Brighton University, Brighton, East Sussex, United Kingdom

Highly Active Anti-Retroviral Therapy (HAART), a combination of nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), and protease inhibitors (PI), has proved very successful in reducing morbidity and mortality associated with HIV infection. However, this extension of lifespan has revealed side effects associated with HAART including cardiovascular dysfunction with HIV patients on HAART becoming the fastest growing population with cardiovascular disease. Poly (ADP-ribose) polymerase (PARP) activation has been found to be a central mediator of endothelial cell dysfunction via reduction of cellular levels of high-energy phosphates and NAD.

The aim of this study was to examine whether the NRTI d4T, NNRT Efavirenz, or the PI Ritnovir activate PARP in endothelial or cardiac cells causing cell dysfunction.

Thoracic aorta isolated from male Sprague-Dawley rats (180-220g) was cut into rings and exposed to the HAART drugs ex vivo for 4 or 6h at 1-10μM. Following this incubation the aortic rings were mounted in organ baths filled with warmed and oxygenated Krebs solution with isometric tension measured with isometric transducers and digitized using PowerLab. The contractile response (to phenylepherine) and relaxant response (to acetylcholine) was determined. Endothelial or heart (H9c2) cell viability was determined using the MTT assay following exposure to HAART drugs for 24 or 48h. PARP activation was determined directly by measuring tritiated NAD incorporation into proteins and indirectly using pharmacological inhibitors.

Following exposure to HAART drugs endothelial cell dysfunction as assessed by acetylcholine-mediated relaxation of aortic rings was found to be disrupted with the RTI potency being d4T>Efavirenz>Ritnovir. Exposure to 10μM HAART drugs for 4h significantly increased the concentration of acetylcholine required for 50% relaxation from 3x10-8M to 2x10-6M, 1x10-6M, and 1.5x10-7M for d4t, Efavirenz and Ritnovir respectively (p<0.05 by analysis of variance with Bonferroni’s correction vs. vehicle treated). The PARP inhibitor PJ-34 (3μM) protected endothelial cell dysfunction form HAART-mediated dysfunction significantly reducing the acetylcholine concentration required for 50% relaxation to 2x10-7M, 1.5x10-7M, 3x10-8M for d4t, Efavirenz and Ritnovir respectively (p<0.05 vs. HAART drugs alone). d4T, Efavirenz and Ritnovir all reduce endothelial and heart cell viability dose (1-30μM) and time (24 and 48h) dependently, effects which are reversed by the simultaneous application of the PARP inhibitor PJ-34. Exposure of endothelial and heart cells to all HAART drugs at 10μM for 4h significantly increases cellular PARP activity; d4T increasing PARP activity by 40% and 80%, Efavirenz increasing activity by 26% and 60% and Ritnovir by 18% and 56% following 4 or 24h exposure respectively (p<0.05 by ANOVA).

In conclusion, HAART drug-induced cardiovascular cell dysfunction appears to be mediated by over-activation of PARP and the subsequent decrease in cellular levels of NAD, NADPH and ATP. Therefore PARP inhibition may be suitable as an adjuvant therapy to HAART to reduce the cardiovascular side effects associated with HAART.