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To the Editors: HIV-1 infection is associated with several immunological disturbances, including a reduction in circulating CD34 hematopoietic progenitor cells. Depletion of CD34 cells may have far-reaching consequences, given their role in immune reconstitution and their association with vascular repair and cardiovascular disease. Although CD34 cells are generally resistant to HIV-1 infection, uninfected CD34 cells from patients with AIDS demonstrate a commitment to apoptosis. This adverse effect is likely due in large part to direct cytotoxic interactions with HIV-1 and, in particular, the envelope glycoprotein gp120. The advent of highly active antiretroviral therapy (HAART) in the treatment of HIV-1 infection has dramatically slowed the rate of progression of HIV-1 to AIDS and improved patient outcomes. As HIV-1–infected patients are living longer, HIV-1–related metabolic and cardiovascular complications are growing in this population, which may be linked to numerical or functional deficits in circulating progenitors. Treatment with protease inhibitors (PIs), an important component of many HAART regimens, generally results in improved viability of CD34 cells from HIV-1– infected individuals. However, this may be largely driven by the antiviral activity of these medications, although the direct effects on CD34 cells are less clear. One prior investigation reported that ritonavir (RTV) had antiapoptotic effects on CD34 cells. Accordingly, we hypothesized that other common PIs would also reduce CD34 cell apoptosis at physiological concentrations (ie, typical maximum plasma levels) in vitro. Freshly isolated CD34 cells from healthy G-CSF–treated human donors (n = 3) were obtained from an independent supplier (Allcells, LLC, Berkeley, CA). Donors tested negative for HIV-1, hepatitis B, and hepatitis C. Viability of cells determined by trypan blue exclusion tests was 95.7% 6 0.4%. Purity, as assessed by flow cytometric analysis with propidium iodide, was 97.7% 6 0.4%. CD34 cells were cultured in serumfree medium (StemCell Technologies, Inc, Vancouver, Canada) supplemented with 100 ng/mL of thrombopoietin, Flt3 ligand, and stem cell factor. Cells were treated for 48 hours with 1 mg/mL RTV, 5.2 mg/mL atazanavir (ATV), or 9.8 mg/mL lopinavir (LPV) (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Positive control experiments were performed using 100 ng/mL HIV-1Bal gp120 (R5 trophic) (AIDS Research and Reference Reagent Program) and HIV-1Lav gp120 (X4 trophic) 12 (Protein Sciences Corporation, Meriden, CT). Activation of caspase-3 was induced in CD34 cells by incubating with staurosporine (1 mmol/L for 3 hours; Sigma Aldrich, St Louis, MO). The concentration of active caspase-3 large subunit in cell lysates was determined using enzyme immunoassays (R&D Systems, Minneapolis, MN). The intraand interassay coefficients of variation for this assay in our laboratory are <10%. Experimental points were performed in duplicate with 3 independent experiments. Differences between treatments were determined by analysis of variance. Where indicated by a significant F value, post hoc tests, with Bonferroni correction for multiple comparisons, were performed to determine differences between treatment groups. Results are expressed as mean 6 standard error of the mean. Statistical significance was set at P < 0.05. On stimulation with staurosporine, both R5 (6.0 6 0.4 ng/mL) and X4 (6.0 6 0.1 ng/mL) gp120 resulted in a 65% greater activation of caspase-3 compared with the control condition (3.6 6 0.4 ng/ mL; P < 0.05) (Fig. 1A). Within the PI group, the capacity of staurosporine to induce an apoptotic response was 40% higher in cells treated with ATV (4.9 6 0.7 ng/mL) and LPV (5.1 6 0.2 ng/mL) compared with the control condition (P < 0.05) (Fig. 1B). In contrast, treatment with RTV (4.1 6 0.5 ng/mL) did not significantly alter intracellular active caspase-3. The novel findings of the present study are that the PIs, ATV and LPV, reduce the resistance of CD34 cells to an apoptotic stimulus. To our knowledge, this is the first study to demonstrate the proapoptotic effects of PIs on CD34 cells from healthy adults. CD34 cells from patients with AIDS demonstrate a marked predisposition toward apoptosis. Zauli et al showed that virus isolates from HIV-1– seropositive patients induced a dosedependent inhibition on the growth of CD34 cells in vitro, an effect blocked by pretreatment with an anti-gp120 antibody. Subsequent investigations further implicated gp120 (in concentrations ranging from 0.01 to 20 mg/mL) in the induction of CD34 cell apoptosis. Our results extend these findings to broadly physiological concentrations of gp120 from 2 strains of the HIV-1 virus, HIV-1Bal and HIV-1Lav, and add to the growing evidence base that indicates a direct cytotoxic activity of HIV-1 gpl20 on human CD34 cells. HAART has been shown to restore CD34 cell function in HIV-1–infected patients. For example, increased CD34 cell colony formation has been documented after 3–6 months of HAART. In addition, the PI RTV lowered apoptosis in CD34 cells from HIV-1–infected patients in vitro but only at a concentration of 5 nmol/L; no differences were recorded at concentrations between 5 and 20 nmol and a decrease was observed at concentrations >20 nmol. Interestingly, these effects have only been documented in CD34 cells from HIV-1–infected patients and only after short-term HAART administration. Our results show that the PIs ATV and LPV reduce resistance to apoptosis in CD34 cells from healthy adults. Although RTV had no effect in the present investigation, it should be noted that the concentration of RTV used in this study was that which is achieved in vivo to boost LPV levels through inhibition of cytochrome P450

Effects of HIV-1 gp120 and Protease Inhibitors on Apoptotic Susceptibility of CD34+ Hematopoietic Progenitor Cells