Liver Fibrosis is Associated with Increased Oxidative Stress in HIV/HCV Co-Infected Adults

1. Jayaweera DT, Campa A, Casillas VJ, Martinez SS, Shin DH, Li Y, Young S, Baum MK Liver Fibrosis is Associated with Increased Oxidative Stress in HIV/HCV Co-Infected Adults

2. Background • Patients co-infected with HIV/HCV comparedto those infected withHCV alone have: • Higher levelsof HCV-RNA. • Anaccelerated progression to liverdisease. • More rapiddevelopment of liver fibrosisand cirrhosis. • Greatermorbidity and mortality.1-6 1. Kim A, et al., 2009. 2.Rotman Y et al., 2009. 3. Pascual-Pareja J et al., 2009 4.Sulkowski MS. 2003. 5.Monga HK et al., 2001. 6. Maciás et al., 2009.

3. Background • HCV infection characterized by oxidative stress generated by chronic inflammation.7,8 • Limited data available on HIV/HCV co-infection. • HIV and HCV independently regulate hepatic fibrosis progression through generation of reactive oxygen species (ROS).9 • HCV core protein localizes to mitochondria and its expression causes inhibition of electron transport, ROS production & glutathione.10 7. Yadav D et al, 20028. Choi J & Ou J, 2006 9. Lin W et al,. 2011 10. Korenaga M. et al., 2005

4. Background • As HCV disease progresses: • Lipid peroxidation products are and glutathione is in serum, PBMC & liver specimens.11,12 • Antioxidant micronutrients are depleted in serum and liver biopsy specimens.8 • Decreased liver micronutrient antioxidant levels are associated with increased liver fibrosis.8 11. Baum MK et al., 201112. Mahmood S et al 2004.

5. Background • Oxidative stress is determined by levels of malondialdehyde (MDA), glutathione, 17,18 & the extent of mtDNA damage.22 • MDA, an index of lipid peroxidation, is in liver, serum, erythrocytes and PBMC in liver diseases,19correlates with HCV disease activity18& fibrosis staging.17,20 • MDA is inversely correlated with hepatic and plasma reduced glutathione.21 • 17. De Maria N et al.,1996. 18. Ko WS et al., 2005. 19. Malvy DJM, et al. 1994 . • 20. Serejo F, et al., 2003. 21. BarbaroG, et al., 1999. 22. Fromenty B et al., 1997.

6. Background • Glutathione is the most abundant non-enzymatic antioxidant in cells.22 • Reduced glutathione (GSH) determines the extent of oxidative capacity. • Oxidized glutathione (GSSG) with severity of HCV, indicating increased oxidative stress & decreased antioxidative capacity.23 22.Akerboom TP et al., 1981. 23. Wu G et al., 2004.

7. Background • Oxidative damage of mtDNA leads to base modification, misreading during replication, and point mutations.24 • The most common form of mtDNAoxidative damage, is increase in the amount of mt-specific 8-oxo-dG.25 • Mt-specific 8-oxo-dG strongly correlates with markers of systemic oxidative stress26-29 & is a risk factor for hepatocellular carcinoma.30 24. Fromenty B et al., 1997. 25. Dagon T et al., 2002. 26. Gerschenson M et al., 2009. Aukurst P et al., 1999. 28. Wu G et al., 2004. 29. Barbaro G et al., 1999. 30. Chuma M et al, 2008.

8. Preliminary Study • HIV/HCV-co-infected compared to HIV-mono-infected participants had: • Higher plasma MDA (1.897±0.835 vs. 1.344±0.223 nmol/mL, p=0.006). • Lower plasma antioxidant concentrations: • Vitamin A (39.5±14.1 vs. 52.4±16.2 mg/dL, p=0.0004). • Vitamin E (8.29±2.1 vs. 9.89±4.5 mg/mL, p=0.043). • Zinc (0.61±0.14 vs. 0.67±0.15mg/L, p=0.016).30 30. Baum MK et al., 2011

9. Preliminary Study • Significantly increased glutathione peroxidase concentration as liver disease advanced, as measured by APRI (β=50.00118; p=0.0082) and FIB-4 (β=0.0029; p=0.0177). • Vitamin A concentration significantly decreased (β=0.00581, p=0.0417) as APRI increased.30 30. Baum MK et al., 2011

10. Objective • To assess the association between oxidative stress and liver fibrosis in HIV/HCV co-infected adults. • To compare markers of oxidative stress between HIV/HCV co-infected and HIV mono-infected adults.

11. Methods • Study Design: • Cross-sectional • Cohorts: -35 HIV/HCV co-infected patients who had recently undergone a liver biopsy, & -101 HIV mono-infected patients • Exclusion Criteria • HBV disease • End stage liver disease • Pregnancy • Chronic Inflammatory Diseases • Morbid obesity • <40 years old • >60 years old • Uncontrolled viral load

12. Methods • Blood was collected for measures of oxidative stress, HIV and HCV disease progression: • Mitochondrial specific 8-hydroxy-deoxyguanosine (mt-specific 8-oxo-dG) • Malondialdehyde(MDA) • Reduced, oxidized and total glutathione • CD4 cell count and HIV viral load • HCV genotype and HCV viral load

13. Methods • Liver fibrosis and inflammation were assessed with liver biopsy and scored using the Metavir Liver Fibrosis Scale. • Liver fibrosis(0 to 4) • 0 = no fibrosis • 1 = portal fibrosis without septa • 2 = few septa • 3 =numerous septa without cirrhosis • 4 = cirrhosis31 • Liver activity • A0 = no histological activity • A1 = mild activity • A2 = moderate activity • A3 = severe activity32 31. The French METAVIR Cooperative Study Group. 1994. 32. BedossaP, et al., 1996.

14. Results Table1: Characteristics of the Population

15. Results • Table 2: Frequencies of Liver Fibrosis and Activity Scores

16. Results (N=136) Table 3: Difference In Mean Measures Of Oxidative Stress Between HIV Mono-Infected And HV/HCV Co-Infected Participants *Statistically significant, p<0.05

17. Results • Low Fibrosis scores were significantly associated with lower levels of 8-oxo-dG (β=0.391, p=0.05,) thanhigh fibrosis scores in HIV/HCV Co-Infection p=0.05 β=0.391 8-oxo-dG (∆Ct) Fibrosis • The grade of inflammation was positively and strongly • associated with oxidized (GSSG) glutathione • (β=0.0034, p=0.002).

18. Results • Using linear regression, HCV viral load was positively associated with 8-oxo-dG (β=1.18, p=0.042) after controlling for age, race, and BMI. Relationship between HCV Viral Load and 8-oxo-dG 8-oxo-dG (∆Ct)

19. Results • Using linear regression, HCV viral load was positively associated with MDA (β=3.97, p=0.049) after controlling for BMI. Relationship between HCV Viral Load and MDA

20. Discussion/Conclusions • HIV/HCV co-infection in our study was associated with pro-oxidative environment; higher oxidative stress correlated with higher fibrosis & inflammation scores, and higher HCV viral load. • Oxidative stress induces proliferation of hepatic stellate cells, TGF-β and collagen synthesis.9, 31, 33 • Oxidative stress participates in the development of progression to fibrosis in patients with Hepatitis C.31, 32, 33 31. Leonardo A, et al, 2004 32. Moriya K, et al, 200133. Choi J & Ou JH, 2006

21. Discussion/ Conclusions • Evidence suggests that antioxidants effectively attenuate the oxidative liver injury, & improve inflammation & fibrosis progression in HCV: • Normalization of liver enzymes, histological improvement & reduction of HCV viral load was observed in a significant proportion of patients with chronic HCV using a combination antioxidant therapy.34,35 • However, antioxidant resveratrol enhanced the HCV replication in vitro. 36 • Strategies to limit HIV/HCV induction of oxidative stress are warranted to slow hepatic fibrosis. Antioxidants need to be tested for their effects on HCV replication. 34. Melhem A, et al, 2005. 35. Gabbay E, et al., 2007 36. Nakamura M, et al, 2010

22. Collaborators • Florida International University • Marianna K Baum, PhD • Adriana Campa, PhD • Dong-Ho Shin, PhD • Sabrina Sales Martinez, MS • Yinghui Li, MS • University of Miami • Dushyantha T Jayaweera, MD • Victor J Casillas, MD • Funded by NIDA grant # R01DA023405 & D-CFAR, University of Miami The project was supported by Award Number R01DA023405 from the National Institute On Drug Abuse. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Drug Abuse or the National Institutes of Health.