Treatment interruption study  suggests that discontinuing combination antiretroviral therapy (cART) is associated with an increase in cardiovascular events in HIV-infected patients. In this population, higher levels of the inflammatory markers high-sensitivity C-reactive protein (hsCRP) and interleukin-6 are associated with a greater risk of cardiovascular disease . These findings suggest that untreated HIV infection, perhaps through increased systemic inflammation, contributes to cardiovascular disease.
We previously demonstrated that the anti-inflammatory drug salsalate improved in-vivo endothelial function in HIV-infected persons not receiving cART . However, salsalate usage in this study was associated with hepatotoxicity. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has been shown to reduce inflammation . On cellular levels, PTX blocks tumor necrosis factor-alpha (TNFα) production in monocytes by inhibiting nuclear factor-kappa B . At doses of 1200 mg/day, PTX reduces TNFα expression and serum triglycerides in patients with AIDS .
Therefore, we performed a prospective, open-label, single-arm pilot study of PTX 400 mg orally thrice daily. Brachial artery flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NTGMD) were measured according to established guidelines  at baseline and after 4 and 8 weeks of PTX. Additional plasma and serum samples for exploratory inflammatory, metabolic, and endothelial activation markers of interest were obtained after a 12 h fast on the mornings of the study visits. PTX levels were measured using a custom in-house modified liquid chromatography–mass spectrometry (MS)/MS assay. Changes in FMD and laboratories from baseline to weeks 4 or 8 were assessed using the Wilcoxon signed-rank test. All tests were two-sided with P values less than 0.05 considered significant.
All participants were at least 18 years of age, had documented HIV infection with a CD4 cell count of at least 350 cells/μl within 1 month of screening, and had been free of antiretroviral treatment for at least 6 months prior to screening. Participants were excluded for known vascular disease, diabetes, hypertension, proinflammatory conditions (apart from hepatitis B or C coinfection), or history of malignancy; estimated creatinine clearance below 50 ml/min, hemoglobin below 9.0 g/dl, alanine aminotransferase or aspartate aminotransferase above three times upper limit normal, or total bilirubin above 2.5 times upper limit normal at screening; fever or recent treatment of infection at screening or any main study visit; or receipt of other anti-inflammatory, investigational or lipid-lowering drugs within 28 days prior to screening. This study was approved by the Clarian (Indiana University School of Medicine) Institutional Review Board. All participants provided written, informed consent.
Of the nine participants who enrolled in the trial [median age 40 (interquartile range or IQR 23–57) years, six men, three non-Hispanic black, median CD4 cell count 552 (IQR 356–645) cells/μl, median HIV-1 RNA 4.5 (IQR 2.7–5.7) log10 copies/ml, four active smokers, median BMI 20.6 (IQR 17.3–26.5) kg/m2, none with hepatitis B, and two with hepatitis C coinfections], one participant was removed at week 2 for initiation of a steroid inhaler for bronchitis and an additional participant was removed at week 5 for initiation of cART. Mild diarrhea was experienced by six participants, but in none was this treatment limiting. No laboratory or clinical toxicities, including graded liver abnormalities, occurred during the study.
The individual changes in FMD with PTX in this trial are shown in Fig. 1(a). The median (range) baseline FMD was impaired at 2.1% (1.3–5.1%) when compared with values in healthy volunteers . As shown in Fig. 1(b), FMD improved significantly from baseline after 4 weeks [N = 8; absolute median (range) increase, 1.5% (−0.7 to 4.9); P = 0.04] and at 8 weeks [N = 7; absolute median (range) increase, 4.4% (2.8–6.5%); P = 0.02]. The median (range) baseline NTGMD was 20.8% (3.9–34.1%), which did not change during the trial.
Median plasma PTX concentrations at weeks 4 and 8 were 73 ng/ml (270 nmol/l) and 97 ng/ml (340 nmol/l), respectively. No concentration–response relationship between plasma PTX levels and FMD was observed. Blood pressure, lipid fractions, homeostatic model assessment-insulin resistance levels, and HIV-1 viral loads did not significantly change with PTX.
We then measured changes in the levels of circulating serum biomarkers [using a custom multiplex assay SearchLight (Endogen) Human Analytes; Rockford, Illinois, USA] reflecting endothelial activation [soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1, and E-selectin)] and inflammation [hsCRP, monocyte chemoattractant protein-1, soluble TNF receptor 1, soluble TNF receptor 2, macrophage inflammatory protein 1-beta, interferon-gamma (IFNγ)-induced protein (IP-10), and TNFα] in the seven participants who completed the trial. As shown in Fig. 1(c and d), only circulating levels of IP-10 and sVCAM-1 decreased significantly (P < 0.05 for both) with use of PTX. As expected, circulating TNFα levels were below the limit of detection at entry and at trial completion in six of these seven relatively healthy individuals with high CD4 cell counts.
Eight weeks of treatment with PTX safely and significantly improved endothelial function in HIV-infected patients not receiving cART. The absolute improvement of 4.4% in FMD that we observed has been associated with a reduced risk of future cardiovascular events in the general population [9,10]. If a similar relationship holds for HIV-infected patients, then PTX could potentially reduce cardiovascular events in this high-risk population. Because of the lack of a control group, we cannot exclude the possibility of a ‘regression to the mean’ effect. However, the magnitudes of improvement in FMD were relatively uniform (Fig. 1a), suggesting that the results were not dependent on the severity of initial endothelial dysfunction.
PTX significantly reduced levels of circulating sVCAM-1 and IP-10. These two molecules are produced by endothelial cells to promote emigration and adhesion of leukocytes as part of the normal vascular inflammatory process and are implicated in the development of atherosclerosis [11,12]. We have previously demonstrated in an endothelial cell culture model that PTX, at concentrations similar to those achieved in our clinical trial, reduced TNFα, IFNγ, and HIV TAT-stimulated endothelial gene expression of VCAM-1 and IP-10 . These in-vitro results strongly corroborate our in-vivo findings. However, we cannot formally exclude the possibility that these biomarker results were not due to effects of changes in alcohol usage on the liver, as we did not assess for alcohol ingestion during this trial.
Randomized, placebo-controlled trials are now underway to investigate the effects of PTX on in-vivo endothelial function both in HIV-infected patients not requiring cART (ClinicalTrials.gov identifier NCT00796822) as well as in those initiating cART (NCT00864916). If these trials confirm that PTX improves in-vivo endothelial function, then PTX may provide an inexpensive and safe modality to reduce the risk of future cardiovascular events in the HIV-infected population.
This research was supported by grants from the National Institutes of Health (K23 HL073682, R01 HL095149, R01 HL072711, and M01 RR00750) and the Indiana University Research Support Funds Program.
We thank the study participants for their generosity and contributions to this study. We also thank Mr Jeffrey Waltz, RDCS, for performance of the brachial artery reactivity studies and also Dr Chul Kim and Mr Brian Taylor for assistance in preparation of the manuscript.
This study was designed and coordinated by S.K.G., R.M.J., K.J.M., M.C., J.R., and M.P.D. Analysis and interpretation of data were done by S.K.G., R.M.J., J.R., C.S., M.C., and M.P.D. Data were collected by S.K.G., R.M.J., and Z.D. The manuscript was drafted by S.K.G. with critical revision by R.M.J., J.R., M.C., and M.P.D.
There are no conflicts of interest.
This work was presented at the 15th Conferences on Retroviruses and Opportunistic Infections (Boston, Massachusetts, USA; 2008).
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