title: HIV associated lipodystrophy: Study of Molecular Mechanisms And Genetic Susceptibility
Creators: name: Majid, Rana
Publisher: name: University of Liverpool Repository
Year: 2016

description: HIV associated lipodystrophy (HIVLD), an adverse effect of combination antiretroviral therapy (cART), further introduces complexity in the management of HIV infection. There is also increased of cardiovascular disease in HIV patients, even in the absence of HIVLD. cART impairs adipogenesis and dysregulates the secretion of adipokines, fat and glucose metabolism in the adipose tissue. This is seen with both protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors. The aim of this thesis was to examine the pathogenesis of cART-induced metabolic disturbances using in vitro models (adipocytes) and in vivo (gene association) studies. Plasma levels of IL18, an adipokine associated with insulin resistance, are elevated in HIVLD patients. The murine 3T3-F442A cell line was used as an in vitro model to assess the effects of PIs (lopinavir [LPV], ritonavir [RTV], atazanavir [ATV]) and NNRTIs (efavirenz [EFV]) in the presence or absence of telmisartan (TEL; 1-5μM). All antiretrovirals (ARV) resulted in a dose dependent increase in IL18 protein levels by ELISA (LPV, 330pg/ml+2.1 [p=0.008]; ATV, 267.3+5.1 [p=0.0001]; RTV, 265.7+2.2 [p=0.0005] and EFV, 98.6+2.4 [p=0.0004]) as compared to the vehicle control; this also correlated with IL18 gene expression determined by RT-PCR. Sequenom MALDI-TOF was used to genotype 14 single nucleotide polymorphisms (SNPs) in the IL18 gene in ARV-treated patients with (HIVLD+; n=115) and without LD (HIVLD-; n=51), but no association was identified. ARV treatment also resulted in the upregulation of NFATC4 gene expression (LPV, 1.9+0.05 [p=0.0015]; ATV, 2.1+0.1 [p=0.0007]; RTV, 1.4+0.05[ p=0.0002] and EFV, 1.8+0.85 [p=0.0001]) as compared to the vehicle. Co-incubation with TEL partially attenuated ARV-mediated upregulation of IL18 (1.6+0.2 [p=0.01) and NFATc4 (1.2+0.1 [p=0.0001]). siRNA knockdown of NFATc4 in adipocytes resulted in significant upregulation in the levels of adiponectin and PPARgamma and down-regulation in IL6 secretion levels following treatment with ARVs. The data suggest that ARV-induced upregulation of NFATc4 could be a mediator of ARV-induced adipocyte toxicity and potentially insulin sensitivity. This study also observed changes in the regulation of various miRNAs following ARV treatment. Validation experiments confirmed LPV to result insignificant downregulation of miR-103 (0.5+ 0.01[p=0.002]) and miR-107 (0.4+ 0.03[p=0.001]) along with their target gene, Cav-1 (0.6+ 0.01[p=0.01]) during adipogenesis; this was reversed by co-incubation with Tel. A systematic review of the genetic associations with HIVLD was also undertaken – this identified that polymorphisms in mitochondrial DNA and cytokines had been the primary ones studied using a candidate gene approach. The results showed that several SNP associations have been identified which are biologically plausible, but in almost all cases, replication of the findings in different cohorts has either been contradictory or limited. This may be due to several reasons including small sample sizes, different diagnostic criteria used for HIVLD, differences in genetic strategy, ethnic variation in genetic architecture and differences in drugs used across different cohorts. Although further studies in larger patient groups with HIVLD should be undertaken, this may not be possible given its rarity now in clinical practice, and perhaps the emphasis should switch to insulin resistance per se. In summary, PIs and NNRTIs upregulate proinflammatory cytokines with NFATc4 potentially playing a major role in their transcriptional upregulation. Differential regulation of specific miRNAs were also found to be important in ARV-mediated dysregulation ofadipogenesis; further validation work which evaluates their role in adipocyte toxicity in vitro and in vivo is now warranted. Whilst this study did not find any genetic association with HIVLD, future studies using larger sample sizes and better defined phenotypes are now required.