HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages (original) (raw)
HIV protease inhibitors induce THP-1 cells and human PBMCs to accumulate cholesteryl esters. HIV protease inhibitors are thought to influence the development of cardiovascular disease primarily through increasing plasma triglycerides and plasma cholesterol levels (7). However, the direct effects of HIV protease inhibitors on macrophages have not been examined in detail. Because one of the prominent features of atherosclerotic lesions is the presence of lipid-laden macrophages (28), we determined whether HIV protease inhibitors could directly affect the amount of cellular cholesterol or cholesteryl esters associated with THP-1 cells, a model human monocyte/macrophage cell line (20), and with isolated human PBMCs. The cells were incubated in the presence of 30 ng/ml of amprenavir, indinavir, ritonavir, or vehicle for 24 hours along with 50 μg/ml of aggregated LDL (an exogenous cholesterol source), and then the total amounts of cholesterol and cholesteryl esters associated with the cells were determined by gas chromatography (29). Figure 1a demonstrates that only ritonavir treatment caused a significant increase in cellular free cholesterol. In contrast, Figure 1b illustrates that compared with the vehicle control (0.01% ethanol), each of the protease inhibitors significantly increased the amount of cell-associated cholesteryl ester. Interestingly, the increase in the amount of cell-associated cholesteryl ester correlated with the degree of dyslipidemia affiliated with the drugs, with amprenavir causing the least increase, followed by indinavir and then ritonavir (1, 30).
HIV protease inhibitors induce the accumulation of cholesteryl ester in THP-1 macrophages and human PBMCs. The human monocyte/macrophage cell line, THP-1, was cultured in 100 nM PMA for 72 hours to promote attachment and differentiation of the cells to a macrophage phenotype. In addition, we used freshly isolated and cultured human PBMCs (37) in these studies. The cells were incubated in the presence of 10% serum and 50 μg/ml of aggregated LDL along with 30 ng/ml of amprenavir, indinavir, ritonavir, or vehicle (ethanol) for 24 hours. The cells were lysed, lipids extracted, and processed to quantify total cellular cholesterol (a) or total cellular cholesteryl ester (b) by gas chromatography. Bars represent mean ± SE, n = 4 with triplicate measurements. *P < 0.01 compared with vehicle, #P < 0.01 compared with amprenavir, +P < 0.01 compared with indinavir. (c) THP-1 cells were lysed and 20 μg of protein was resolved by SDS-PAGE and immunoblotted with antibodies for CD36, SRA, and actin. Cross-reactive material was visualized by chemiluminescence. The exposure time was 2 minutes. The data are representative of five independent experiments. Essentially identical immunoblots were generated with human PBMCs (data not shown).
CD36 plays a role in HIV protease inhibitor–induced sterol accumulation. Multiple mechanisms can alter cellular cholesteryl ester levels, including new sterol synthesis and uptake of exogenous sterol from lipoproteins. To test whether HIV protease inhibitors were inducing new sterol synthesis, THP-1 cells were treated with vehicle or 30 ng/ml of amprenavir, indinavir, or ritonavir in the presence of [3H]acetate (31). The cells were then processed to isolate sterols and the amount of new sterol synthesized was determined by TLC (29). Each of the treatment groups synthesized 42,000 ± 2,401 disintegrations per minute (dpm) sterol per mg cell protein per 24 hours, indicating that global sterol synthesis was not altered. We next determined whether HIV protease inhibitors altered the levels of CD36 and SRA, two receptors involved in the uptake of exogenous lipoproteins and the formation of foam cells (15). THP-1 cells were treated as described above and analyzed by SDS-PAGE and immunoblot. We found that CD36 protein levels were increased in the presence of the HIV protease inhibitors, whereas the levels of SRA were not affected (Figure 1c). In addition, the relative increase in the level of CD36 (with amprenavir, 3.4-fold; indinavir, 6.2-fold; ritonavir, 13.1-fold) approximated the measured increase in cholesteryl ester accumulation (compare Figure1b to Figure 1c). The actin immunoblots indicate that equivalent amounts of protein were applied to each lane.
To determine whether the increase in CD36 protein levels played a role in the increase in cholesteryl ester accumulation, CD36-blocking antibodies and a CD36 morpholino (which blocks transcription) were used. Figure 2a demonstrates that the CD36 antibody cross-reacts with a 90-kDa protein in CHO cells transfected with cDNA encoding human CD36 but not with CHO cells transfected with vector only. THP-1 cells contain the same 90-kDa cross-reacting band, and importantly, THP-1 cells treated with the CD36 morpholino did not produce the 90-kDa band. These data demonstrate that the CD36-blocking antibody recognizes human CD36 and that treatment with a CD36 morpholino decreases CD36 protein below the level of detection. THP-1 cells were then incubated in the presence of 30 ng/ml of amprenavir, indinavir, or ritonavir for 24 hours along with CD36-blocking antibodies (20 μg/ml), nonrelevant isotype–matched antibodies (20 μg/ml), or a CD36 morpholino (25 nmol). At the end of the incubation period, the total amount of cholesterol and cholesteryl esters associated with the cells was determined by gas chromatography. Figure 2b demonstrates that the nonrelevant IgM antibody did not affect the ability of the protease inhibitors to induce sterol accumulation. However, both the CD36-blocking antibody and the CD36 morpholino completely inhibited the ability of the protease inhibitors to induce sterol accumulation.
CD36 blocking antibodies and a CD36 morpholino prevent the accumulation of sterol. (a) THP-1 cells were cultured in 100 nM PMA for 72 hours to promote attachment and differentiation of the cells to a macrophage phenotype and then treated with 25 nmol of a CD36 morpholino for 24 hours. The THP-1 cells and CHO cells expressing human CD36 (hCD36) or the vector only were lysed and 20 μg of protein was resolved by SDS-PAGE and immunoblotted with the CD36 blocking antibody. Cross-reactive material was visualized by chemiluminescence. The exposure time was 2 minutes. The data are representative of three independent experiments. (b) THP-1 cells were cultured in 100 nM PMA for 72 hours to promote attachment and differentiation of the cells to a macrophage phenotype. The cells were incubated in the presence of 10% serum and 50 μg/ml of aggregated LDL, along with 30 ng/ml of amprenavir, indinavir, ritonavir, or vehicle (ethanol) for 24 hours. In addition, different sets of cells also contained one of the following: 20 μg/ml of CD36 blocking IgM, 20 μg/ml nonrelevant IgM, or a CD36 morpholino (25 nmol). After the treatment period, the cells were lysed, lipids extracted, and processed to quantify total cellular cholesterol and cholesteryl esters by gas chromatography. Bars represent mean ± SE, n = 5 with triplicate measurements. *P < 0.01 compared with vehicle, +P < 0.01 compared with amprenavir, #P < 0.01 compared with indinavir.
The use of blocking CD36 antibodies and a CD36 morpholino are pharmacological approaches to demonstrate the involvement of CD36 in HIV protease–induced increases in macrophage cholesteryl ester. To be certain that an increase in CD36 protein levels is required for the increase in macrophage cholesteryl esters, we isolated peritoneal macrophages from various mouse models and then treated the isolated cells with HIV protease inhibitors as described above. After the treatment period the cells were processed and the amount of cholesterol and cholesteryl ester associated with the cells was quantified. Figure 3 demonstrates that treatment with vehicle (0.01% ethanol) did not alter the level of sterol in macrophages isolated from any of the mouse strains. Macrophages isolated from C57BL/6, apoE null, and LDLR null mice all accumulated sterol in response to treatment with ritonavir. In contrast, macrophages isolated from mice lacking CD36 (CD36 null and apoE/CD36 null mice) did not accumulate sterol in the presence of HIV protease inhibitors. These data strongly support the conclusion that CD36 was required for HIV protease inhibitor–induced increases in macrophage sterol levels.
Ritonavir does not induce sterol accumulation in peritoneal macrophages isolated from CD36 null mice. Peritoneal macrophages were isolated from the indicated mouse strains and then incubated in the presence of 10% serum and 50 μg/ml of aggregated LDL, along with 30 ng/ml of ritonavir or vehicle (0.01% ethanol) for 24 hours. After the treatment period the cells were lysed, lipids extracted, and processed to quantify total cellular cholesterol and cholesteryl esters by gas chromatography. Bars represent mean ± SE, n = 3 with triplicate measurements. *P < 0.01 compared with vehicle.
HIV protease inhibitors promote the generation of lipid-laden macrophages independent of dyslipidemia. The in vitro data established that HIV protease inhibitors increase the cellular level of macrophage cholesteryl esters in a CD36-dependent manner. These data implied that frank dyslipidemia is not necessary to generate lipid-laden macrophages and subsequently atherosclerotic lesions. To directly test this prediction, two doses of amprenavir, indinavir, or ritonavir were given via the drinking water to LDLR null mice for 0, 4, or 8 weeks. The LDLR null mice were maintained on a normal chow diet, thereby greatly alleviating diet-induced dyslipidemia and diet-induced atherosclerotic lesion formation. The lower doses of protease inhibitors did not alter plasma triglyceride (Figure 4a) or cholesterol/cholesteryl ester (Figure 4b) levels, whereas the higher doses of inhibitors induced a pronounced increase in plasma triglyceride and cholesterol/cholesteryl ester levels. These data demonstrate that we established doses of HIV protease inhibitors that promote dyslipidemia and doses that do not promote dyslipidemia, in the same animal model.
Effect of HIV protease inhibitors on plasma lipids. Six-week-old male LDLR null mice on a chow diet were given vehicle control (0.01% ethanol) or the following protease inhibitors in their drinking water: amprenavir (23 or 75 μg/mouse/day), indinavir (25 or 75 μg/mouse/day), or ritonavir (10 or 50 μg/mouse/day). The total plasma triglyceride (a) and cholesterol/cholesteryl ester (b) levels were determined after 0, 4, and 8 weeks of treatment with a commercial kit (Wako Chemicals USA Inc.) or gas chromatography. Bars represent mean ± SE, n = 8. *P < 0.01 compared with vehicle. White bars are vehicle controls, black bars are low-dose protease inhibitors, and gray bars are high-dose protease inhibitors.
We next determined whether HIV protease inhibitors altered the level of cholesterol/cholesteryl ester associated with macrophages in vivo by isolating peritoneal macrophages (23) from the same group of mice described above and quantifying the level of cholesterol/cholesteryl ester by gas chromatography (29). All three protease inhibitors induced an increase in the level of peritoneal macrophage sterol compared with vehicle-treated animals (Figure 5). Importantly, the lower doses of protease inhibitors caused a dramatic increase in macrophage sterol levels without a concomitant increase in plasma lipids (Figure 4), whereas the higher doses increased plasma lipids (Figure 4) and further increased macrophage sterol levels.
HIV protease inhibitors induce an increase in peritoneal macrophage cholesterol/cholesteryl ester levels. Six-week-old male LDLR null mice on a chow diet were given vehicle control (0.01% ethanol) or the following protease inhibitors in their drinking water: amprenavir (23 or 75 μg/mouse/day), indinavir (25 or 75 μg/mouse/day), or ritonavir (10 or 50 μg/mouse/day). After 8 weeks of treatment, peritoneal macrophages were isolated and cholesterol/cholesteryl ester mass was determined by gas chromatography. Bars represent mean ± SE, n = 8. *P < 0.01 compared with vehicle, #P < 0.01 compared with low dose of the same protease inhibitor.
The in vitro data demonstrated that HIV protease inhibitors increased the expression of CD36 in THP-1 cells. To determine whether HIV protease inhibitors stimulated an increase in the level of macrophage CD36 protein in vivo, we isolated peritoneal macrophages from LDLR null mice, generated cell lysates, and analyzed the proteins by SDS-PAGE and immunoblotting. Figure 6a demonstrates that HIV protease inhibitors did not alter the expression of SRA or actin. However, the inhibitors dramatically increased the amount of CD36 both at the low and high doses of inhibitor. In addition, the relative increase in the level of CD36 (with amprenavir, 3.1-fold; indinavir, 5.3-fold; ritonavir, 12.9-fold) approximated the measured increase in cholesteryl ester accumulation (see Figure 5). We next determined whether the HIV protease inhibitors specifically affected macrophage CD36 protein levels or whether they also increased CD36 protein levels in other tissues. We isolated cardiac myocytes, adipocytes, and platelets and analyzed the levels of CD36 by immunoblot. HIV protease inhibitors did not alter the levels of CD36 in cardiac myocytes, adipocytes, or platelets, suggesting that these compounds specifically alter CD36 levels in macrophages (Figure 6b).
HIV protease inhibitors induce an increase of CD36 protein levels in peritoneal macrophages. Six-week-old male LDLR null mice on a chow diet were given vehicle control (0.01% ethanol) or the following protease inhibitors in their drinking water: amprenavir (23 or 75 μg/mouse/day), indinavir (25 or 75 μg/mouse/day), or ritonavir (10 or 50 μg/mouse/day). (a) After 8 weeks of treatment, peritoneal macrophages were isolated, the cells were lysed, and 20 μg of protein was resolved by SDS-PAGE and immunoblotted with antibodies for CD36, SRA, and actin. The data are representative of eight mice. (b) After 8 weeks of treatment, cardiac myocytes (38), adipocytes (39), and platelets (40) were isolated, the cells were lysed, and 20 μg of protein was resolved by SDS-PAGE and immunoblotted with antibodies against CD36. The data are representative of eight mice.
Because HIV protease inhibitors induced macrophages to accumulate sterol, we next determined whether HIV protease inhibitors promoted the formation of atherosclerotic lesions. To directly determine this, two doses of amprenavir, indinavir, or ritonavir were given in the drinking water to LDLR null mice for 8 weeks. At the conclusion of the study, the ascending and descending aortas were removed and opened, and the areas covered by lesions were quantified by image analysis (26, 27). All of the animals treated with HIV protease inhibitors had significantly greater lesion area than did vehicle-treated animals (Figure 7). Animals treated with amprenavir had the smallest increase in lesion area and those treated with ritonavir had the largest increase in lesion area, consistent with the relative increase observed for CD36 and sterol levels in peritoneal macrophages. In addition, the higher doses of protease inhibitors caused an additional increase in lesion area compared with lower doses of the same protease inhibitors.
HIV protease inhibitors induced the formation of atherosclerotic lesions in LDLR null mice. Six-week-old male LDLR null mice on a chow diet were given vehicle control (0.01% ethanol) or the following protease inhibitors in their drinking water: amprenavir (23 or 75 μg/mouse/day), indinavir (25 or 75 μg/mouse/day), or ritonavir (10 or 50 μg/mouse/day). These mice were then used to quantify the surface area of atherosclerotic lesions. To quantify lesions, we first removed the aorta from the arch to the ileal bifurcation and dissected away extraneous tissue. The intimal surfaces were exposed by a longitudinal cut. The aortas were placed under a dissecting microscope equipped with a video camera attachment that captures the image in a computer file. Atherosclerotic lesions on the intimal aortic surface appear as bright white areas compared with the thin and translucent aorta. Areas of intima covered by atherosclerotic lesions were quantified with ImagePro software. This software analyzes differences in contrast to identify areas covered by lesions. Bars represent mean ± SE, n = 8. *P < 0.01 compared with vehicle, #P < 0.01 compared with low dose of the same protease inhibitor.
The data suggested that HIV protease inhibitors induced the upregulation of macrophage CD36, which then promoted the formation of atherosclerotic lesions independent of dyslipidemia. To directly test whether the increase in CD36 was responsible for the increase in atherosclerotic lesion formation, apoE null and apoE × CD36 double null mice were fed a chow diet and given vehicle or ritonavir (10 μg/mouse/day) in their drinking water for 6 weeks. The dose of ritonavir used did not induce dyslipidemia greater than that seen in vehicle-treated mice (data not shown). At the conclusion of the study, atherosclerotic lesion area was quantified in the ascending and descending aortas by image analysis (Figure 8). Vehicle-treated apoE null and apoE × CD36 double null mice had relatively small lesions, and importantly, the extent of the lesions was similar in both types of animals. Ritonavir-treated apoE null mice had a substantial increase in lesion area compared with vehicle-treated control mice. In contrast, ritonavir did not induce atherosclerotic lesions in apoE × CD36 double null mice.
Ritonavir did not induce formation of atherosclerotic lesions in apoE × CD36 null mice. Six-week-old male apoE null and apoE × CD36 double null mice were fed a chow diet and given vehicle (0.01% ethanol) or ritonavir (10 μg/mouse/day) in their drinking water for 6 weeks. At the conclusion of the study the extent of atherosclerotic lesions was quantified as above. Data are presented as mean ± SE. n = 12. *P < 0.01 compared with vehicle.
HIV protease inhibitors increase CD36 and PPAR-γ mRNA in a PKC-dependent manner. Previous studies have demonstrated that CD36 expression can be upregulated through activation of the transcription factor PPAR-γ (32). In addition, earlier studies have demonstrated that PKC is involved in the pathway responsible for the upregulation of CD36 expression (33). To determine whether ritonavir induced an increase in CD36 and PPAR-γ expression in a manner dependent on PKC, we treated human PBMCs as indicated and analyzed the amount of CD36 and PPAR-γ mRNA by Northern blot. Figure 9 demonstrates that ritonavir or aggregated LDL by themselves did not alter CD36 or PPAR-γ mRNA levels; however, the combination of ritonavir and aggregated LDL increased CD36 mRNA levels by 22-fold and PPAR-γ levels by 18-fold. Diacylglycerol (DAG) (20 μM) and PMA (100 nM), activators of PKC, stimulated an increase in CD36 and PPAR-γ mRNA independent of ritonavir. Importantly, two different PKC inhibitors, Go6976 (500 nM) and calphostin C (200 nM), inhibited the ability of ritonavir to increase CD36 and PPAR-γ mRNA. Finally, two different PPAR-γ activators, 15d-PGJ2 (1 μM) and ciglitazone (13 μM), stimulated the expression of CD36 and PPAR-γ mRNA, and the increase in expression was blocked by PKC inhibitors.
Ritonavir increases CD36 and PPAR-γ mRNA in a PKC-dependent manner. Human PBMCs were incubated in the presence of 10% serum and 50 μg/ml aggregated LDL (agLDL) and/or 30 ng/ml ritonavir as indicated for 24 hours. In addition, some sets of cells were also treated with PMA (100 nM), (diacylglycerol [DAG]; 20 μM), Go6976 (500 nM), calphostin C (200 nM), 15d-PGJ2 (1 μM), or ciglitazone (13 μM). The cells were then lysed, mRNA was isolated, and the relative amounts of CD36, PPAR-γ, and GAPDH mRNA were determined by Northern analysis. Shown are representative data from three independent experiments.