A Pilot Study of Urinary Markers of Endothelial Function & Oxidant Stress for the Prediction of Cardiovascular Disease Risk with Antiretroviral therapy Michael S. Boger 1, Ginger Milne 2, Husamettin Erdem 1, Valerie Mitchell 1, David W. Haas 1, Jason Morrow 2, Todd Hulgan 1 Divisions of 1 Infectious Diseases and 2 Clinical Pharmacology Department of Medicine Vanderbilt University School of Medicine

Introduction Potent antiretroviral therapy (ART) dramatically improves morbidity & mortality from HIV infection ART may increase cardiovascular disease (CVD) risk Friis-Moller N et al. NEJM 2003; 349: DAD Study Group. NEJM 2007; 356: Traditional Framingham prediction may underestimate CVD risk in HIV infected patients on ART Law MG et al. HIV Med 2006; 7: De Socio GV et al. J Infect 2008; 57: 33-40

Introduction Inflammation, oxidant stress, & endothelial dysfunction are central to the pathogenesis of atherosclerosis/CVD Deakin S et al. Atheroscler Thromb Vasc Biol 2007; 27: HIV-infection &/or ART may influence these factors hsCRP correlates with CVD outcomes in the general population; limited data in HIV infection Pearson TA et al. Circulation 2003; 107: A urinary marker (isoprostane) of oxidant stress correlated with traditional CVD risk factors Wang B et al. Atherosclerosis 2006; 184: Basarici I et al. Coron Artery Dis 2007; 18: Vascular reactivity appears impaired in HIV infection Torriani F et al. 4 th IAS Conference Abstr WEAB302 Solages A et al. Clin Infect Dis 2006; 42:

Introduction Eicosanoids are involved in inflammation, endothelial function, & oxidant stress Urinary assays for eicosanoids are noninvasive, precise, accurate, & reproducible Milne GL et al. J Biol Chem 2005; 280: We suspect that quantifying eicosanoids may help assess cardiovascular risk in this population

Eicosanoids: Inflammation, Endothelial Function, & Oxidant Stress Membrane Phospholipids Arachidonic Acid COX Prostaglandin-H 2 ThromboxanesProstacyclinProstaglandins Isoprostanes Autoxidation (Platelets)(Endothelium)(Smooth Muscle) 8-ISO-PGF2α (PGF2α) 11 DTxB2 (TxB2) 2,3 DN-6KETO (PGI-M) PGE-M Khanapure SP et al. Curr Top Med Chem 2007; 7: Vasoconstriction Platelet aggregation Oxidative tissue damage Vasoconstriction Platelet activation Chemotaxis Vasodilation Inhibits platelet aggregation Inhibits vascular smooth muscle proliferation Vasoconstriction Vascular smooth muscle proliferation

Eicosanoids: Inflammation, Endothelial Function, & Oxidant Stress Membrane Phospholipids Arachidonic Acid COX Prostaglandin-H 2 ThromboxanesProstacyclin Isoprostanes Autoxidation (Platelets)(Endothelium)(Smooth Muscle) 8-ISO-PGF2α (PGF2α) 11 DTxB2 (TxB2) 2,3 DN-6KETO (PGI-M) PGE-M Khanapure SP et al. Curr Top Med Chem 2007; 7: Prostaglandins

Research Hypotheses & Aim Hypotheses ART metabolic effects include increased eicosanoid production These biomarkers are coupled with inflammation, oxidant stress, & endothelial dysfunction Aim To correlate novel markers of inflammation, oxidant stress, & endothelial function with serum lipids & hsCRP in HIV infected patients on ART

Study Population HIV infection > 12 mos On ART including ≥ 2 NRTIs HIV RNA < 400 copies/mL No CAD or DM No lactic acidosis No malignancy No aspirin use No tobacco use

Methods & Statistical Analyses Cross-sectional pilot study of a prospective cohort Urine eicosanoids quantified by liquid chromatography/mass spectroscopy Spearman’s correlation Wilcoxon rank-sum

Characteristics of Study Subjects (N=33) Age in years, median (IQR)45 (39-51) Female sex, n (%)8 (24%) Non-white race, n (%)18 (55%) BMI, median (IQR)26 (24-27) hsCRP mg/dL, median (IQR)2.2 ( ) CD4 cells/mm 3, median (IQR)515 ( ) HIV RNA copies/mL, median (range) Non-HDL mg/dL, median (IQR) Lipoatrophy, n (%) 50 (50-470) 124 ( ) 9 (27%) PI-based ART, n (%) Intermittent NSAID use 15 (45%) 20 (55%)

Results: Urinary Eicosanoids Eicosanoid* (ng/mg cr) Reference 1-4 Study Population PGF2α1.60 ± ± 1.40 TxB20.37 ± ± 0.17 PGI-M0.14 ± ± 0.06 PGE-MM: ± 1.50 F: 6.00 ± 0.70 M: ± F: 6.57 ± 5.39 Morrow JD et al. J Chromatogr 1993; 612: Murphey LJ et al. Anal Biochem 2004; 334: Daniel VC et al. J Chromatogr 1994; 653: Morales CR et al. Clin Chim Acta 2001; 314: 93-9 * Data are mean ± 1 SD

Results: Gender Differences in Urinary Eicosanoids PGF2α* (ng/mg cr) Total (N= 33) Men (N=25) Women (N =8) Overall1.9 ( )1.8 ( )2.0 ( ) PI use Yes No 1.4 ( ) 2.2 ( ) 1.4 ( ) 1.9 ( ) 1.3 ( ) 2.3 ( ) Lipoatrophy Yes No 2.3 ( ) 1.8 ( ) 2.3 ( ) 1.6 ( ) 1.6 ( ) 2.0 ( ) *Data are median (IQR) No association with age, race, CD4, HIV RNA, NSAID use, BMI p=0.04p=0.07

Results: Gender Differences in Urinary Eicosanoids PGI-M* (ng/mg cr) Total (N= 33) Men (N=25) Women (N =8) Overall0.12 ( ) 0.10 ( ) PI use Yes No 0.11 ( ) 0.12 ( ) 0.12 ( ) 0.12 ( ) 0.07 ( ) 0.13 ( ) Lipoatrophy Yes No 0.15 ( ) 0.11 ( ) 0.15 ( ) 0.11 ( ) 0.11 ( ) 0.10 ( ) *Data are median (IQR) No association with age, race, CD4, HIV RNA, NSAID use, BMI p=0.07

Results: Correlation of Urinary Eicosanoids & hsCRP Eicosanoid*Total (N= 33) Men (N=25) Women (N =8) PGF2α0.36, , , 0.49 TxB20.33, , , 0.01 PGI-M0.39, , , 0.02 PGE-M0.26, , , 0.54 *Data are correlation coefficient, p-value

Results: Correlation of Urinary Eicosanoids & Non-HDL Cholesterol Eicosanoid*Total (N= 33) Men (N=25) Women (N =8) PGF2α0.02, , , 0.65 TxB20.38, , , 0.80 PGI-M0.19, , , 0.15 PGE-M0.23, , , 0.76 *Data are correlation coefficient, p-value

Summary Minimally invasive, reliable methods to predict metabolic problems of ART are needed Urinary eicosanoids may be promising markers of CVD health in HIV patients on ART These markers of inflammation, oxidant stress & endothelial function correlated with non-HDL cholesterol & hsCRP Sex-specific associations of urinary eicosanoids with traditional CVD risk factors were seen Larger studies including CVD outcomes are needed to confirm & expand our findings

Acknowledgements Jason Morrow, MD

Acknowledgements Persons with HIV infection who volunteered for this study NIH/NCCAM Career Development Award K23 AT Vanderbilt CTSA grant 1UL1RR (NCRR/NIH) Vanderbilt-Meharry CFAR P30 AI54999 NIH Molecular Basis of Infectious Diseases Training Program T32 AI Vanderbilt University GCRC Vanderbilt Human Analytical Isoprostane Core Facility

A Pilot Study of Urinary Markers of Endothelial Function & Oxidant Stress for the Prediction of Cardiovascular Disease Risk with Antiretroviral therapy Michael S. Boger 1, Ginger Milne 2, Husamettin Erdem 1, Valerie Mitchell 1, David W. Haas 1, Jason Morrow 2, Todd Hulgan 1 Divisions of 1 Infectious Diseases and 2 Clinical Pharmacology Department of Medicine Vanderbilt University School of Medicine