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Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag(240-249) TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.

In addition to inhibiting cholesterol biosynthesis, statins also inhibit the formation of isoprenoid intermediates, which are required for the activation of the Rho/Rho kinase (ROCK) pathway. Increased ROCK activity has been implicated in causing endothelial dysfunction and atherosclerosis. However, it is not known whether statins, at doses used to lower cholesterol levels, inhibit ROCK activity in humans with atherosclerosis. Furthermore, it is not known whether lipophilic and hydrophilic statins differ in their ability to inhibit ROCK activity. Accordingly, we enrolled 30 men with stable atherosclerosis (low-density lipoprotein [LDL] > or =100 mg/dL) in a randomized, double-blind study comparing equivalent LDL-lowering doses of a hydrophilic statin (rosuvastatin 10 mg once a day) with a lipophilic statin (atorvastatin 40 mg once a day) for 28 days. We assessed the change in lipids, ROCK activity, and flow-mediated dilation (FMD) of the brachial artery before and after statin therapy. Both treatment groups exhibited comparable 30% to 32% and 42% to 45% reductions in total and LDL cholesterol, respectively. Only atorvastatin reduced triglycerides, and neither statin altered high-density lipoprotein cholesterol. Whereas both statins inhibited ROCK activity (p <0.0001), the extent of inhibition was greater with rosuvastatin (18 +/- 2% vs 8 +/- 2%, p = 0.0006). Statins also improved FMD from 7.4 +/- 0.6 to 9.3 +/- 0.4 (p = 0.003) with rosuvastatin being slightly better than atorvastatin. The inhibition of ROCK activity by statins did not correlate with reductions in LDL (p = 0.57) but was associated with improvement in FMD. In conclusion, these findings provide direct clinical evidence that statins, at clinically relevant doses, could differentially inhibit ROCK activity and improve endothelial function by cholesterol-independent mechanism.

BACKGROUND

In addition to inhibiting cholesterol synthesis, statins (HMG-CoA reductase inhibitors) decrease the formation of isoprenoid intermediates required for the activation of key signaling pathways, including Rho/Rho kinase (ROCK). In experimental settings, statins inhibit ROCK and reverse vascular dysfunctions in atherosclerosis, independent of cholesterol reduction. It is not known whether statins inhibit ROCK activity in humans with atherosclerosis.

METHODS

We investigated 35 patients with stable atherosclerosis in a randomized, double-blind study comparing treatment with high-dose (80mg/d) or low-dose (10mg/d) atorvastatin to placebo for 28 days. Blood samples for leukocyte ROCK activity, fasting lipids, and high-sensitivity C-reactive protein (hs-CRP) were obtained on days 0, 7, 14, and 28 after randomization and change over time with the two statin treatments relative to placebo was examined.

RESULTS

Atorvastatin 80mg/d reduced ROCK activity (p=0.002 vs. placebo). This decline was rapid and significant within 2 weeks of treatment. The inhibition of ROCK by atorvastatin (80mg/d) remained significant even after controlling for changes in low-density lipoprotein cholesterol (LDL-C) and triglycerides (p=0.01). Furthermore, there was no correlation between changes in ROCK activity and changes in LDL-C (r=0.2, p=0.25) or triglycerides (r=0.1, p=0.55). There was a modest correlation between ROCK inhibition and change in hs-CRP among patients randomized to atorvastatin 80mg/d (r=0.6, p=0.07).

CONCLUSIONS

These first-in-man findings demonstrate that high-dose atorvastatin rapidly inhibits the pro-atherogenic Rho/ROCK pathway, independent of cholesterol reduction. This inhibition may contribute to the clinical benefits of statins. Rho/ROCK may provide a useful therapeutic target in patients with atherosclerosis.