impaired lipoprotein processing in hiv patients on...

1714

HIV–infected patients receiving antiretroviral therapy (ART) present with a dyslipidemia and insulin

resistance,1 which places them at a high risk for accelerated cardiovascular disease.2–6 The increased cardiovascular disease among HIV/ART patients is a persistent public health challenge for which current therapies are not adequate. HIV/ART dyslipidemia comprises hypertriglyceridemia, low plasma high-density lipoprotein (HDL) cholesterol (HDL-C), and elevated plasma non–HDL-C concentrations.7,8 Other distinctive pathogenic features of HIV/ART dyslipidemia include hypoadiponectinemia,9,10 increased lipolysis,11,12 and hepatic free fatty acids flux, which increases very-low-density lipoprotein (VLDL) synthesis,13 in a way that is

mechanistically linked to defects in HDL metabolism.8,14 Given that the low plasma HDL-C concentrations place HIV/ART patients at a high cardiovascular disease risk, we tested the hypothesis that the compositions and properties of HDL from these patients were distinct from control subjects in a way that impaired HDL metabolism. This hypothesis was tested by studying dyslipidemic HIV/ART patients with and without hypertriglyceridemia, and control non-HIV normolipidemic (NL) subjects.

Materials and MethodsMaterials and Methods are available in the online-only Supplement.

© 2013 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.113.301538

July

90

Objective—HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional.

Approach and Results—Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001).

Conclusions—Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness. (Arterioscler Thromb Vasc Biol. 2013;33:1714-1721.)

Key Words: hepatocyte cholesteryl ester uptake ◼ high-density lipoprotein function ◼ HIV dyslipidemia ◼ lipoprotein composition

Received on: February 5, 2013; final version accepted on: April 17, 2013.From the Section of Atherosclerosis and Vascular Medicine (B.K.G., J.L.R., H.J.P.) and Diabetes Research Center, Division of Diabetes, Endocrinology,

and Metabolism (R.R.-E., D.I., I.C., A.B.), Department of Medicine, Baylor College of Medicine, Houston, TX; and Atherosclerosis & Lipoprotein Research, The Methodist Hospital Research Institute, Houston, TX (B.K.G., H.J.P.).

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301538/-/DC1.Correspondence to Henry J. Pownall, PhD, Atherosclerosis & Lipoprotein Research, The Methodist Hospital Research Institute, 6670 Bertner St, MS

F8-060, Houston, TX 77030. E-mail [email protected]

Impaired Lipoprotein Processing in HIV Patients on Antiretroviral Therapy

Aberrant High-Density Lipoprotein Lipids, Stability, and Function

Baiba K. Gillard, Joe L. Raya, Raul Ruiz-Esponda, Dinakar Iyer, Ivonne Coraza, Ashok Balasubramanyam, Henry J. Pownall

by guest on June 3, 2018http://atvb.ahajournals.org/

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http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301538/-/DC1

mailto:[email protected]

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Gillard et al Reduced HDL Functionality in HIV/ART 1715

Results

Altered Plasma Lipid Profiles in HIV/ARTLipoprotein compositions were determined in a large subset of HIV patients from the Heart Positive Study15 (predominantly those who completed the 24-week trial). Subset plasma lipid profiles simulated those of the entire Heart Positive patient set (Table I in the online-only Data Supplement). Treatment groups were 1, placebo; 2, diet and exercise (D&E); 3, fibrate+D&E; 4, niacin+D&E; 5, fibrate+niacin+D&E. In all 5 treatment groups, patient plasma lipid profiles at baseline satisfied the lipid criteria for metabolic syndrome, that is, hypertriglyceridemia and a low plasma HDL-C concentration. Moreover, the HDL-C levels were lower and those for TG were higher than those of NL controls. Analysis of variance confirmed that the lipid profiles at baseline for the 5 groups, assigned at random on enrollment in the Heart Positive Study, were not significantly different. As reported for the entire Heart Positive study16 treatment with fenofibrate+D&E (group 3), niacin+D&E (group 4), and the combined therapy of fenofibrate+niacin+D&E (group 5) significantly reduced plasma TG, with median reductions of 35% to 42%. Treatments including niacin with or without fenofibrate increased median HDL-C by 21% to 24%. Treatments including fibrate with or without niacin reduced median non–HDL-C by 20% to 25%.

Altered Lipoprotein Composition in HIV/ARTCompared with NL controls, low-density lipoprotein (LDL)-%TG values of Heart Positive HIV/ART patients at baseline were a median 2-fold higher, whereas other constituents were not significantly different (Table 1). HDL compositions were also significantly different, with higher %TG, % cholesteryl ester (CE), and % free cholesterol (core components), and lower % phospholipid and % protein (surface components) in Heart Positive HIV/ART patients versus NL controls. The fraction of HDL composed of surface and core components, respectively, were lower and higher in HIV/ART patients with significant elevations of both the %CE and the %TG. The elevation of neutral lipid core components suggested that hypertriglyceridemic HIV/ART HDL is larger than NL HDL.

Effect of Treatment on Lipoprotein CompositionThe 5 treatment arms differentially affected HDL and LDL neutral lipid compositions. As expected, the placebo group showed no change in HDL and LDL composition (Figure 1; group 1). The various treatments did not have much effect on lipoprotein CE content (Figure 1A and 1C). Although increased for all treatments, median LDL %CE (average increase of 4%) was not significantly different except for group 2 (D&E). Similarly, although all treatments trended toward decreased HDL %CE (average 6%), this was not significant. In contrast, all 4 interventions reduced the median LDL %TG contents (Figure 1B) and HDL %TG (Figure 1D). Because of the limited sample size and large individual variability in %TG values, the differences in paired entry versus after-therapy values for any single treatment group only approached P<0.05 significance. However, for all treatments combined there was a 24% decrease in LDL %TG (P=0.003) and a 25% decrease in HDL %TG (P=0.004). These results indicate that whereas the elevated TG contents of both LDL and HDL were responsive to treatment, the elevated CE content of HTG-HIV/ART HDL was more resistant to these treatment protocols.

Hypertriglyceridemic HIV/ART HDL Are not Stable on FreezingAs the neutral core to surface lipid ratio of lipoproteins increases, the particle size increases.17 Assessment of lipoprotein particle size by size exclusion chromatography (SEC) revealed that Heart Positive HIV/ART HDL were unstable on freezing and exhibited other SEC peaks eluting earlier (larger particle size), which were absent from the chromatograms of normal HDL (Figure 2A). We assigned these large species to HDL fusion.18 As shown in Figure 2B, SEC uncovers freezing-dependent differences in the profile of NL control HDL after multiple freeze–thaw cycles. However, the effect of freeze–thaw is much more profound for the HDL from 4 Heart Positive HIV/ART patients. Thus, the frozen-while-stored samples could not be used for studies of HDL sizing or functionality.

HDL Composition of Non-HTG HIV/ARTHIV/ART HDL from a second HIV/ART patient group were stored at 4°C. The plasma lipid values for HIV/ART

Table 1. Heart Positive HIV and NL Control HDL and LDL Compositions at Baseline*

Donor n % Protein % PL % FC % CE % TG % (CE+TG)

High-density lipoprotein composition

HIV 114 42.0 (36.6–45.7) 19.3 (18.4–20.7) 4.08 (3.36–5.25) 27.2 (24.2–30.0) 7.41 (5.68–10.17) 35.3 (31.2–38.3)

NL 12 47.7 (45.3–50.7) 23.1 (21.6–24.6) 2.08 (1.92–2.41) 21.8 (18.9–23.7) 4.56 (3.21–6.19) 26.6 (24.7–29.0)

P† <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Ratio 0.9 0.8 2.0 1.3 1.6 1.3

Low density lipoprotein composition

HIV 113 24.2 (22.4–26.1) 18.7 (17.8–19.7) 7.56 (6.86–8.33) 36.5 (33.4–39.1) 11.97 (9.27–16.3) 49.2 (47.6–50.9)

NL‡ 22 22 8 42 6 48

Ratio 1.1 0.9 0.9 0.9 2.0 1.0

CE indicates cholesteryl ester; FC, free cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NL, normolipidemic; PL, phospholipid; and TG, triglyceride.*Values are median and (25%–75%) range.†Mann–Whitney rank-sum test P value for comparison of Heart Positive HIV with NL values.‡NL values for LDL composition are from Havel et al.38


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1716 Arterioscler Thromb Vasc Biol July 2013

patients are compared with those of NL controls and the Heart Positive HIV/ART patients in Table 2. Unlike the Heart Positive Clinical Trial, which had completed by this time, the second HIV/ART patient group was not selected for hypertriglyceridemia. According to their plasma lipid concentrations, these patients presented with isolated low plasma HDL-C (P=0.004 versus NL controls) and reduced total plasma cholesterol (P=0.025 versus NL controls) but no hypertriglyceridemia. The HDL compositions for this group (Table 3) show that HDL-CE contents were lower than those of NL controls, the opposite of what was observed for the HTG HIV/ART patients. However, similar to the hypertriglyceridemic Heart Positive patients, this group of normotriglyceridemic HIV/ART patients also had HDL neutral lipid cores enriched in TG relative to CE compared

with NL control HDL. HDL samples from this group were studied further for stability, size, and functionality.

HIV/ART HDL Are Less Stable to Chaotropic Perturbation Than NL Control HDLIncubation of HDL with guanidinium chloride (GdmCl) induces the release of lipid-free apolipoprotein (apo)AI and the formation of a larger fused HDL (Figure 3).18,19 Comparison of the effects of GdmCl on the lipoprotein profiles of HDL from NL controls and HIV/ART patients revealed notable differences in the amounts of the released products (Figure 3A and 3B). Although GdmCl converted the HDL from both NL controls and HIV/ART patients to the expected products, the relative amounts of fused HDL and lipid-free apoAI formed were different (Figure 3C and 3D). Post-GdmCl, the relative amount of HDL remaining was lower and the amount of lipid-free apoAI was higher in HIV/ART HDL versus NL control HDL. Accordingly, even non-HTG HIV/ART HDL is less stable than NL HDL.

HIV/ART Lipoproteins Are Larger Than NL Control LipoproteinsThe high neutral lipid content of LDL and HDL of Heart Positive patients suggested these might be larger lipoproteins.17

D&EFibrateNiacin

Group

LDL

% C

E

15202530354045

EntryCompletion

LDL

% T

G

0

10

20

30

40

HD

L %

CE

15

20

25

30

35

40

HD

L %

TG

0

5

10

15

20

25

- + + + + - - + - + - - - + + 1 2 3 4 5

A

B

C

D

0.05

0.06 0.06

0.05 0.06

Figure 1. Comparison of the effects of 4 different antilipidemic therapies vs placebo control on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) composition. LDL and HDL % cholesteryl ester (CE) and % triglyceride (TG) weight composition are shown as median, box plot of (25%–75%) range and bar (10%–90%) range at entry (white bars) and after therapy (gray bars). Therapy groups 1 to 5 are denoted on the abscissa. Wilcoxon signed rank-sum analysis of paired entry and after-therapy values are indicated for P<0.06. For all treatments combined, both LDL and HDL %TG decrease significantly: P=0.003 and 0.004, respectively.

HIV/ART HDL Post Freeze-Thaw

Abs

orba

nce 28

0 nm

Elution Volume, mL

15 20 25 30 35

A

B

C

D

NL HDL Post Freeze-Thaw

C Post Freeze Thaw 2

Elution Volume, mL

15 20 25 30 35

Abs

orba

nce 28

0 nm

B Post Freeze-Thaw 1

A NL HDL

D Post Freeze-Thaw 3

Figure 2. Effects of freezing on high-density lipoprotein (HDL) as assessed by size exclusion chromatography. Top, A–D, Four different HIV/antiretroviral therapy (ART) HDL samples after 3 freeze–thaw cycles. Bottom, A–D, a single normolipidemic (NL) control HDL sample analyzed before freezing and after 1 to 3 freeze–thaw cycles.


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The sizes of lipoproteins in the total lipoproteins (TLP) from non-HTG HIV patients and controls were compared by SEC (Figure II in the online-only Data Supplement). The means±SE of these data, calculated and plotted as shown in Figure 4, reveal profound differences between HIV/ART and NL control TLP. NL control TLP contains prominent peaks for VLDL, LDL, and HDL with relatively small SEs (Figure 4A). SEC profiles for HIV/ART TLP differ (Figure 4B). First, peaks for the 3 major lipoproteins are shifted to earlier elution volumes corresponding to larger particle sizes (compare gray vertical lines). Second (Figure 4B, ×10 insert), the SEC of HIV/ART TLP contains a peak between LDL and VLDL, which we assign to intermediate density lipoprotein (IDL). Last, the SEs at each elution volume of the HIV/ART TLP chromatogram are much larger than those of control TLP. The relative amounts of the lipoproteins are also altered. On the basis of the ratios of the peak heights, HIV/ART lipoproteins are altered relative to NL controls, with higher amounts of VLDL and IDL relative to LDL and HDL (Figure 5). The ratios relative to LDL for NL controls and HIV/ART patients are as follows: VLDL/LDL=1.10 versus 2.10 (P=0.032); IDL/LDL=0.160 versus 0.436 (P=0.001). These larger sizes, and the greater amounts of VLDL and IDL relative to LDL and HDL, suggest delayed processing of apoB lipoproteins in HIV plasma.

Validation of a Functional Assessment of NL Control Versus HIV/ART HDLBecause of the altered CE content and the lower stability of the HIV/ART HDL samples, we hypothesized that HIV/ART HDL are dysfunctional in the final step of reverse cholesterol transport, transfer of HDL CE to hepatocytes. The most straightforward method for measuring hepatocyte HDL-CE uptake requires preparation of HDL-[3H]CE, a procedure that can take 10 days and requires significant quantities of starting HDL20 to yield HDL-[3H]CE with >99% radiochemical purity,21,22 followed by uptake assays according to Acton et al.23 This assay is impractical in the context of large patient numbers and limited plasma volumes, so we modified and validated a competitive assay of hepatic CE uptake that we have used to compare the HDL-[3H]CE uptake before and after remodeling by streptococcal serum-opacity factor.20 This assay uses NL controls and HIV/ART patient HDL to compete with the uptake of a standard stock HDL-[3H]CE (Figure III in the online-only Data Supplement). Inhibition constants, ID

50%,

were calculated from the inhibitor HDL dose–response curves. In this assay, a large ID

50% corresponds to HDL that interacts

poorly with hepatic HDL receptors and vice versa. Our assay is reproducible; each panel in Figure III in the online-only Data Supplement contains assays on the same HDL sample repeated on different days, with ≤17 weeks between assays. Assays in triplicate showed a within-day variability of 10.6% (n=28) and day-to-day variability of 10.4% (n=4).

Table 2. Plasma Lipid Values for HIV Patients With Isolated Low HDL-C (non-HTG HIV) Compared With Heart Positive HIV and NL Controls*

Donor n Cholesterol, mg/dL Triglyceride, mg/dL HDL-C, mg/dL Calculated LDL-C, mg/dL Non–HDL-C, mg/dL

Heart Positive HIV 114 195 (172–230) 248 (171–329) 37 (31–42) 108 (77–137) 158 (136–187)

P† 0.914 <0.001 <0.001 0.755 0.010

Non-HTG HIV 10 125 (72–208) 109 (91–228) 33 (20–58) 44 (34–128) 80 (53–159)

P† 0.025 0.668 0.004 0.052 0.106

NL control 12 196 (166–233) 118 (90–142) 67 (52–92) 103 (90–120) 124 (111–153)

HDL-C indicates high-density lipoprotein cholesterol; HTG, hypertriglyceridemic; LDL-C, low-density lipoprotein cholesterol; and NL, normolipidemic.*Values are median and (25%–75%) range.†Mann–Whitney rank-sum test P value for comparison of HIV values with NL values.

Abso

rban

ce28

0 nm

Control

Elution Volume, mL15 20 25 30 35

2 M GdmCl

NLA

B

C D

HIV/ART

Normal HIV/ART

HD

L Pe

ak H

eigh

t, m

AU28

0 nm

0

2

4

6

8

Post 2 M GuCl

Normal HIV/ART

LF A

po A

-I/H

DL Fu

sed

0

1

2

3

4

5

6

7Post 2 M GuCl

p=0.001 p=0.09

Figure 3. Comparison of the stability of high-density lipoprotein (HDL) from HIV/antiretroviral therapy (ART) and normolipidemic (NL) control subjects. A and B, Examples of size exclusion chromatography profiles of NL and HIV/ART HDL before (gray shaded curve) and after (line) treatment with 2 mol/L guanidinium chloride (GdmCl). Stability was assessed on the basis of the HDL peak height after treatment (C) and the lipid-free apolipoprotein (apo)AI/HDLfused ratio (D). Less fused (lipidated) HDL and more lipid-free apoAI is formed from the HDL of the HIV/ART patients than from that of the NL controls.


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HIV HDL Is a Poor Competitor for Hepatocyte HDL-CE UptakeUsing this competitive inhibition assay, we compared the ID

50%

of HDL from HIV/ART patients with those from NL controls. As shown in Figure 6A, ID

50% for individual NL and HIV/

ART HDL distributed over a wide range, 72 to 211 and 75 to 191 µg/mL HDL protein for NL and HIV/ART, respectively. These ranges, which are far greater than the assay variability, illustrate the individual heterogeneity of donor HDL with respect to selective hepatic CE uptake and the power of this assay to distinguish HDL functionality on the basis of hepatic CE uptake. Our data show that the mean ID

50% for the NL

group was lower than that of the HIV/ART subjects (rank-sum P=0.052), indicating the HIV/ART HDL was a poorer competitor, that is, required more HDL to reach its ID

50%.

Importantly, adjustment of these data for each donor’s HDL-C level, which were lower for the HIV/ART patients (Figure 6B), revealed the dysfunctionality of HIV/ART HDL. The difference between HIV/ART and NL plasma to affect HDL-CE uptake was profound—CE uptake inhibition by HIV/ART HDL, expressed as plasma volume-equivalents, was much lower. NL plasma contains 23.6±1.7 ID

50% units/ mL versus 14.0±1.8

ID50%

units/mL for HIV/ART plasma (P<0.001). The mean

Table 3. HDL Composition of Heart Positive HIV, HIV With Isolated Low HDL-C (Non-HTG HIV), and NL Control Donors*

High-Density Lipoprotein Composition

Donor n % Protein % PL % FC % CE % TG % (CE+TG) TG/(TG+CE)

Heart Positive HIV 114 42.0 (36.6–45.7) 19.3 (18.4–20.7) 4.08 (3.36–5.25) 27.2 (24.2–30.0) 7.41 (5.68–10.17) 35.3 (31.2–38.3) 0.22 (0.16–0.29)

P† <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.076

Non-HTG HIV 10 49.3 (47.1–59.0) 24.8 (18.8–27.4) 2.22 (2.03–2.70) 15.2 (10.8–18.7) 5.34 (3.39–6.70) 20.3 (16.6–24.2) 0.25 (0.17–0.35)

P† 0.156 0.817 0.448 0.006 0.621 0.008 0.049

NL 12 47.7 (45.3–50.7) 23.1 (21.6–24.6) 2.08 (1.92–2.41) 21.8 (18.9–23.7) 4.56 (3.21–6.19) 26.6 (24.7–29.0) 0.17 (0.12–0.25)

CE indicates cholesteryl ester; FC free cholesterol; HDL-C, high-density lipoprotein cholesterol; HTG, hypertriglyceridemic; NL, normolipidemic; PL, phospholipid; and TG, triglyceride.

*Values are median and (25%–75%) range.†Mann–Whitney rank -sum or Student t test P value for comparison of HIV values with NL values.

HIV

Elution Volume, mL15 20 25 30 35

-1

0

1

2

3

x10

ControlA

B

Abs

orba

nce 28

0 nm

0.0

0.3

0.6

0.9 HDL

LDLVLDL

Figure 4. Comparison of lipoprotein sizes by size exclusion chromatography (SEC) of total lipoproteins (TLP) from normolipidemic (NL) controls and HIV/antiretroviral therapy (ART) subjects. Plots in A and B, respectively, are the average SEC profiles of the TLP from 12 NL controls and 10 HIV/ART Subjects. Individual chromatograms are in Figure III in the online-only Data Supplement. The insert in B is a 10× expansion of that region of the chromatogram to better reveal peaks for intermediate-density lipoprotein and low-density lipoprotein (LDL). The black and gray lines represent the mean and SE of the chromatograms. The dashed gray lines demonstrate that the average size of very-low-density lipoprotein (VLDL), LDL, and high-density lipoprotein (HDL) are larger in HIV/ART than in NL subjects.

Normal HIV/ART

VLD

L Peak

Hei

ght/L

DL Pe

ak H

eigh

t

0

1

2

3

4

5

6Rank sum p=0.032A B

C D

Normal HIV/ART

IDL Pe

ak H

eigh

t/LD

L Peak

Hei

ght

0.0

0.2

0.4

0.6

0.8

ttest p=0.001

ttest p=0.002

Normal HIV/ART

IDL Pe

ak H

eigh

t/HD

L Peak

Hei

ght

0.0

0.2

0.4

0.6

Rank sum p=0.027

Normal HIV/ART

VLD

L Peak

Hei

ght/H

DL Pe

ak H

eigh

t

0

1

2

3

4

5

Figure 5. Relative amounts of very-low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) in HIV/antiretroviral therapy (ART) and normolipidemic (NL) control plasma. Peak heights for each lipoprotein were determined from the individual donor total lipoprotein chromatograms shown in Figure II in the online-only Data Supplement. The ratios of VLDL and IDL relative to low-density lipoprotein (LDL) are shown in A and B, and relative to high-density lipoprotein (HDL) in C and D. HIV/ART plasma contains significantly more VLDL (P<0.03) and especially IDL (P<0.002) relative to both LDL and HDL than NL control plasma contains. The ratio of LDL/HDL was not significantly different (data not shown).


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rate of CE uptake from HDL-[3H]CE is inhibited on average twice more effectively by NL HDL than by HIV/ART HDL, when measured at plasma concentrations of the respective HDL. Thus, at plasma concentrations HIV/ART HDL interact with hepatocytes less effectively than NL HDL do.

DiscussionPlasma Lipid Profiles of HIV/ART Patients With HTG or Isolated Low HDL-C Versus NL ControlsThe Heart Positive HIV/ART patients in the 4 therapeutic and placebo groups have similar lipid profiles (Table I in the online-only Data Supplement). The higher LDL- and HDL-TG contents within the HTG HIV/ART group (Table 1) are consistent with the CE transfer protein-mediated transfer of TG from VLDL, which are elevated, to LDL and HDL. This effect is dose dependent with respect to fasting plasma TG levels and is expected to yield less stable TG-rich LDL and HDL.24

TG enrichment may underlie the greater freeze instability of Heart Positive versus control subjects. The effects of the interventions on LDL and HDL compositions were consistent with this model (Figure 1). LDL- and HDL-TG from patients on D&E and receiving niacin or fenofibrate were lower. The plasma lipoprotein profile of the non-HTG HIV/ART patients was characterized by isolated low HDL-C (Table 2), low HDL %CE, and a TG-enriched neutral lipid core (Table 3).

Metabolism of HIV/ART Lipoproteins Is ImpairedAccording to the total lipoprotein (TLP) SEC profiles, the dyslipidemia in HIV/ART patients is highly heterogeneous (large SEs), and is associated with the appearance of lipoproteins that are larger than those of NL controls (Figure 4). Consistent with this, the data of Aragonès et al25 showed greater heterogeneity in HIV HDL and increased CE in the larger HDL subfractions. Two observations suggest that increased lipoprotein heterogeneity is a result of impaired lipoprotein hydrolysis. First, the SEC profiles (Figure 4) showed that all HIV/ART lipoproteins elute earlier than their NL control counterparts; second, these data also show that unlike control TLP, HIV/ART TLP contains a prominent peak for IDL (Figure 4). Increased amounts of VLDL and IDL in HIV/ART fasting plasma indicate delayed processing of these lipoproteins (Figure 5). Given that both IDL- and HDL-TG are substrates for hepatic lipase (HL), which reduces their size by hydrolysis, we hypothesize that HL activity is impaired under the conditions of HIV/ART dyslipidemia, a hypothesis that is supported by reports that HL and to a lesser extent lipoprotein lipase activities are lower in HIV/ART-associated dyslipidemia26; lower lipoprotein lipase activity would also explain the larger HIV/ART versus NL control VLDL. Although endothelial lipase activity has not been reported in HIV samples, low endothelial lipase activity could also cause larger HDL as well as reduced HDL phospholipid,27 but would be expected to cause higher rather than lower plasma HDL-C.28

Interaction of HIV/ART HDL With Hepatocytes Is ImpairedAccording to our competitive assay of HDL-CE uptake by Huh7 cells, differences in the ID

50% of NL control versus HIV/

ART HDL were nearly significant (P=0.052; Figure 6A). Moreover, when the ID

50% data were normalized to the plasma

HDL-C levels of each individual, HIV/ART plasma contained significantly less competitive inhibition activity for hepatocyte HDL-CE uptake than NL plasma did (P<0.001). Decreased interaction of HIV/ART HDL with hepatic receptors may contribute to the observed elevated HDL %CE in the Heart Positive patient population (Table 1). It would suggest that the higher HDL-CE may result from an inability of HIV/ART HDL to deliver its CE to hepatocyte receptors for uptake and clearance. The molecular basis for this is not known but may be a result of altered surface protein structure or configuration in response to an altered core composition.

HDL StabilityHIV/ART HDL were found to be less stable than NL HDL both on freezing and on chaotropic perturbation. Importantly, HDL instability has been uncovered by lecithin:cholesterol acyltransferase, CE transfer protein, phospholipid transfer

Inhibition/mL plasma

Normal HIV/ART

ID50

% E

quiv

alen

ts/m

L Pl

asm

a

0

10

20

30

40 Mean + SE ttest p < 0.001

CE Uptake inhibition/HDL Mass

Normal HIV/ART

ID50

%,

g/m

L H

DL

Prot

ein

µ

0

50

100

150

200

250A

B

Mean + SERank sum p = 0.052

Figure 6. Inhibition of hepatocyte high-density lipoprotein (HDL)-cholesteryl ester (CE) Uptake by HDL from normolipidemic (NL) and HIV/antiretroviral therapy (ART) Subjects. A, ID50% for individual donor HDL indicating inhibitory activity per mg HDL protein. B, Inhibitory potency of each patient’s plasma expressed in terms of the volume of plasma needed to achieve 50% inhibition. Black dots, individual values; gray dot and bars, mean±SE. The combination of low plasma HDL cholesterol (HDL-C) and the higher median ID50% in the HIV/ART vs NL control subjects results in an average ID50% equivalent that is 41% lower for the HIV/ART vs NL subjects, P<0.001.


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protein, and serum-opacity factor, all of which induce fusion or the release of lipid-free apoAI.22,29–32 Although there have been many studies of the effects of GdmCl on HDL,18,19,33,34 this study is the first to show that the effects of GdmCl on HDL vary among dyslipidemic patients and NL controls, and that the NL control and HIV/ART HDL respond differently to this denaturant. According to several studies, HDL resides in a kinetic trap from which it can escape via both physico-chemical18,19 and biological perturbations—lecithin:cholesterol acyltransferase, CE transfer protein, phospholipid transfer protein, and serum-opacity factor.29–31,35 Thus, in the context of the kinetic model of Gursky,18 HIV/ART HDL is less stable than NL control HDL. Given that a major marker of instability is the release of lipid-free apoAI, one could speculate that the plasma factors cited above mediate production of more lipid-free apoAI in HIV/ART patients compared with NL controls. A negative consequence of this would be increased renal clearance of apoAI, which would have the effect of lowering plasma HDL-C levels; this hypothesis needs more rigorous testing.

A Metabolic Model of Dysfunctional Lipoprotein Processing in HIV/ART DyslipidemiaAccording to our data, HIV/ART dyslipidemia is associated with larger HDL, LDL, and VLDL, and impaired HDL hepatocyte binding, which likely affects hepatocyte HDL-CE uptake. Other studies in HIV patients have demonstrated peripheral tissue hyperlipolytic activity11 and lower lipoprotein and HL activities.26,36 In contrast, the initial step in reverse cholesterol transport, macrophage-cholesterol efflux to HDL, is unaltered by HIV and its treatment. Rose et al8 found similar rates of activated RAW 264.7 macrophage-cholesterol efflux to plasma of HIV-infected patients, currently treated HIV-infected patients, and HIV-negative subjects. Taken together, these data support a metabolic model for HIV dyslipidemia (Figure 7) that begins with peripheral tissue hyperlipolytic activity11 that results in the release of higher amounts of free fatty acids that are extracted by the liver and used for the production of more VLDL, thereby producing a hypertriglyceridemic state. In the presence of high VLDL-TG concentrations, CE transfer protein mediates the exchange of VLDL TG for HDL and LDL-CE, thereby producing TG-rich HDL and LDL. CE transfer protein activity has been reported to be elevated8 or within normal limits37 in HIV/ART subjects. Lipoprotein-TG hydrolysis by HL reduces their sizes by removing core lipids, and when this process is inhibited larger TG-rich lipoprotein species are observed. In our study, there is even some residual IDL, which under normolipemic conditions is converted to LDL by HL. All lipoprotein classes from HIV/ART are larger than those from NL subjects suggesting resistance to the activities of hepatic or lipoprotein lipases. As a consequence of the high TG content of the HDL particles, we speculate that they are less stable and that the HIV/ART apoAI is more labile than that of NL HDL. Whether the instability influences function remains to be investigated. Nevertheless, the CE of HIV/ART HDL are hepatically extracted at a lower rate than those of NL HDL because of their intrinsically lower uptake and the lower plasma HDL-C levels in HIV/ART patients.

AcknowledgmentsWe thank Hu Yu Alice Lin for excellent technical assistance, Xaioyuan Perrard for the mRNA analyses, and Lele Li for initial work on high-density lipoprotein stability to guanidinium chloride treatment.

Sources of FundingThis work was supported by National Institutes of Health grants HL73696 (A. Balasubramanyam), RO1-HL-30914 and RO1-HL56865 (H.J. Pownall), and P30DK079638 for support of the Diabetes Research Center at Baylor College of Medicine.

DisclosuresNone.

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Figure 7. Metabolic model for the production of dysfunctional lipoproteins in HIV/ART dyslipidemia. See text for discussion. ATGL indicates adipose triglyceride lipase; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; HDL, high-density lipoprotein; HL, hepatic lipase; HSL, hormone sensitive lipase; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; NEFA, non esterified fatty acids; SRBI, scavenger receptor class B type I; TG, triglyceride; and VLDL, very-low-density lipoprotein.


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10. Vigouroux C, Maachi M, Nguyên TH, Coussieu C, Gharakhanian S, Funahashi T, Matsuzawa Y, Shimomura I, Rozenbaum W, Capeau J, Bastard JP. Serum adipocytokines are related to lipodystrophy and metabolic disorders in HIV-infected men under antiretroviral therapy. AIDS. 2003;17:1503–1511.

11. Sekhar RV, Jahoor F, White AC, Pownall HJ, Visnegarwala F, Rodriguez-Barradas MC, Sharma M, Reeds PJ, Balasubramanyam A. Metabolic basis of HIV-lipodystrophy syndrome. Am J Physiol Endocrinol Metab. 2002;283:E332–E337.

12. Hadigan C, Borgonha S, Rabe J, Young V, Grinspoon S. Increased rates of lipolysis among human immunodeficiency virus-infected men receiving highly active antiretroviral therapy. Metabolism. 2002;51:1143–1147.

13. Reeds DN, Mittendorfer B, Patterson BW, Powderly WG, Yarasheski KE, Klein S. Alterations in lipid kinetics in men with HIV-dyslipidemia. Am J Physiol Endocrinol Metab. 2003;285:E490–E497.

14. Mujawar Z, Rose H, Morrow MP, Pushkarsky T, Dubrovsky L, Mukhamedova N, Fu Y, Dart A, Orenstein JM, Bobryshev YV, Bukrinsky M, Sviridov D. Human immunodeficiency virus impairs reverse cholesterol transport from macrophages. PLoS Biol. 2006;4:e365.

15. Asztalos BF, Tani M, Schaefer EJ. Metabolic and functional relevance of HDL subspecies. Curr Opin Lipidol. 2011;22:176–185.

16. Balasubramanyam A, Coraza I, Smith EO, Scott LW, Patel P, Iyer D, Taylor AA, Giordano TP, Sekhar RV, Clark P, Cuevas-Sanchez E, Kamble S, Ballantyne CM, Pownall HJ. Combination of niacin and fenofibrate with lifestyle changes improves dyslipidemia and hypoadiponectinemia in HIV patients on antiretroviral therapy: results of “heart positive,” a randomized, controlled trial. J Clin Endocrinol Metab. 2011;96:2236–2247.

17. Shen BW, Scanu AM, Kézdy FJ. Structure of human serum lipoproteins inferred from compositional analysis. Proc Natl Acad Sci U S A. 1977;74:837–841.

18. Mehta R, Gantz DL, Gursky O. Human plasma high-density lipoproteins are stabilized by kinetic factors. J Mol Biol. 2003;328:183–192.

19. Pownall HJ, Hosken BD, Gillard BK, Higgins CL, Lin HY, Massey JB. Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning. Biochemistry. 2007;46:7449–7459.

20. Gillard BK, Rosales C, Pillai BK, Lin HY, Courtney HS, Pownall HJ. Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol. Biochemistry. 2010;49:9866–9873.

21. Rosales C, Gillard BK, Courtney HS, Blanco-Vaca F, Pownall HJ. Apolipoprotein modulation of streptococcal serum opacity factor activity against human plasma high-density lipoproteins. Biochemistry. 2009;48:8070–8076.

22. Gillard BK, Courtney HS, Massey JB, Pownall HJ. Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption. Biochemistry. 2007;46:12968–12978.

23. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996;271:518–520.

24. Pownall HJ, Brauchi D, Kilinç C, Osmundsen K, Pao Q, Payton-Ross C, Gotto AM Jr, Ballantyne CM. Correlation of serum triglyceride and its reduction by omega-3 fatty acids with lipid transfer activity and the neutral lipid compositions of high-density and low-density lipoproteins. Atherosclerosis. 1999;143:285–297.

25. Aragonès G, Beltrán-Debón R, Rull A, Rodríguez-Sanabria F, Fernández-Sender L, Camps J, Joven J, Alonso-Villaverde C. Human immunodeficiency virus-infection induces major changes in high-density lipoprotein particle size distribution and composition: the effect of antiretroviral treatment and disease severity. Clin Chem Lab Med. 2010;48:1147–1152.

26. Yarasheski KE, Tebas P, Claxton S, Marin D, Coleman T, Powderly WG, Semenkovich CF. Visceral adiposity, C-peptide levels, and low lipase activities predict HIV-dyslipidemia. Am J Physiol Endocrinol Metab. 2003;285:E899–E905.

27. Singaraja RR, Sivapalaratnam S, Hovingh K, et al. The impact of partial and complete loss-of-function mutations in endothelial lipase on high-density lipoprotein levels and functionality in humans. Circ Cardiovasc Genet. 2013;6:54–62.

28. Edmondson AC, Brown RJ, Kathiresan S, et al. Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans. J Clin Invest. 2009;119:1042–1050.

29. Silver ET, Scraba DG, Ryan RO. Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles. J Biol Chem. 1990;265:22487–22492.

30. Lusa S, Jauhiainen M, Metso J, Somerharju P, Ehnholm C. The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion. Biochem J. 1996;313:275–282.

31. Liang HQ, Rye KA, Barter PJ. Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase. J Lipid Res. 1996;37:1962–1970.

32. Rye KA, Hime NJ, Barter PJ. Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion. J Biol Chem. 1997;272:3953–3960.

33. Nichols AV, Gong EL, Blanche PJ, Forte TM, Anderson DW. Effects of guanidine hydrochloride on human plasma high density lipoproteins. Biochim Biophys Acta. 1976;446:226–239.

34. Rosseneu M, Van Tornout P, Lievens MJ, Schmitz G, Assmann G. Dissociation of apolipoprotein AI from apoprotein-lipid complexes and from high-density lipoproteins. A fluorescence study. Eur J Biochem. 1982;128:455–460.

35. Rosales C, Tang D, Gillard BK, Courtney HS, Pownall HJ. Apolipoprotein E mediates enhanced plasma high-density lipoprotein cholesterol clearance by low-dose streptococcal serum opacity factor via hepatic low-density lipoprotein receptors in vivo. Arterioscler Thromb Vasc Biol. 2011;31:1834–1841.

36. Baril L, Beucler I, Valantin MA, Bruckert E, Bonnefont-Rousselot D, Coutellier A, Caumes E, Katlama C, Bricaire F. Low lipolytic enzyme activity in patients with severe hypertriglyceridemia on highly active antiretroviral therapy. AIDS. 2001;15:415–417.

37. Johnston CS, Kim CM, Buller AJ. Vinegar improves insulin sensitivity to a high-carbohydrate meal in subjects with insulin resistance or type 2 diabetes. Diabetes Care. 2004;27:281–282.

38. Havel RJ, Goldstein JL, Brown MS. Lipoproteins and lipid transport. In: Bondy PK, Rosenberg LE, eds. The Metabolic Control of Disease. Philadephia, PA; Saunders Publishing; 1980:398–494.

Patients with HIV receiving antiretroviral therapy (ART) present with a unique dyslipidemia that places them at risk for cardiovascular disease. We compared HIV/ART patients with normolipidemic subjects. All HIV/ART lipoproteins were larger than the corresponding normolipidemic lipoproteins; the HIV/ART high-density lipoprotein are richer in neutral lipids, less stable, and bind poorly to hepatocytes. These findings were based on new assays of high-density lipoprotein stability, and functionality; this study was the first to apply these assays to a clinical population. These findings are unprecedented in HIV/ART research and show that the underlying mechanisms for the unique HIV/ART dyslipidemia are impaired lipoprotein clearance by plasma lipases or hepatic receptors. This research brings a new perspective on the pathogenesis of HIV/ART-associated dyslipidemia, with high relevance to some other metabolic diseases—Cushing syndrome, polycystic ovarian disease, and obesity-linked diabetes mellitus.

Significance


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Balasubramanyam and Henry J. PownallBaiba K. Gillard, Joe L. Raya, Raul Ruiz-Esponda, Dinakar Iyer, Ivonne Coraza, Ashok

High-Density Lipoprotein Lipids, Stability, and FunctionImpaired Lipoprotein Processing in HIV Patients on Antiretroviral Therapy: Aberrant

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Materials and Methods Study Populations: Our study was based on two HIV/ART patient groups. Initial results were obtained from stored baseline and final plasma from the Heart Positive Study (www.ClinicalTrials.gov ID NCT00246376), a 24-week, randomized, placebo-controlled, double blind test of additive mono and combination therapy for dyslipidemia in HIV patients on stable ART.1 The Heart Positive inclusion criterion was hypertriglyceridemia (fasting plasma TG > 150 mg/dL). Participants were randomized to a placebo (Group 1) and four different therapeutic groups—diet and exercise (D & E) (Group 2), D & E + fenofibrate (Group 3), D & E + niacin (Group 4), and D & E + fibrate + niacin (Group 5). The design of the D&E program has been reported.1, 2 From the total population of 114 patients, 110 completed the 24-week trial and four participated for 12 to 20 weeks. We also recruited NL control patients and a second group of HIV/ART patients with near normal plasma TG levels but low plasma HDL-C levels to study HDL size, stability, and functionality without sample freezing. Plasma lipids were determined in the Atherosclerosis Clinical Research Laboratory. Liver function tests, glucose, and insulin were determined in The Methodist Hospital Pathology Laboratory or by Quest/Labcorp. The study was approved by the Institutional Review Boards of Baylor College of Medicine and Legacy Community Health Center.

Lipoprotein isolation: Lipoproteins were isolated from EDTA plasma by sequential flotation at d=1.006 g/mL (VLDL), d =1.02 g/mL (IDL), d=1.063 g/mL (LDL), and d=1.21 g/mL (HDL); the lipoprotein-deficient fraction (d> 1.21 g/mL) was also collected in the final flotation step.3, 4 Fractions were stored at -80oC until analyzed for lipoprotein composition. Lipoproteins from the second group of HIV patients and normal controls were similarly isolated and stored at 4 °C.

Lipoprotein Compositions: The major lipoprotein-lipids, cholesteryl ester, phospholipids, free cholesterol, and triglyceride were assayed using commercial enzyme-based kits (Wako). Phospholipid assay mixtures contained 10 mM CaCl2 to correct for calcium depletion in the EDTA plasma. Protein was determined by RC DC Protein Assay (Biorad), a valid protein assay even in the presence of KBr. Total Lipoprotein (TLP) Profiles: Lipoproteins sizes were assessed by size exclusion chromatography (SEC) using two Superose HR6 columns (GE Health Care-Life Sciences) in tandem. After flotation at d = 1.21 g/mL at 40,000 rpm (Beckman Ti 50.2) for three days, aliquots (200 µL) were injected and eluted at 0.5 mL/min. The column effluent was monitored by measuring absorbance at 280 nm. The mean ± SE TLP profiles for NL Control and HIV/ART were calculated and plotted in Sigma Plot 10 (Systat Software, Inc.). Based on 14 consecutive injections of a cholesteryl ester-rich microemulsion, the reproducibility of the elution volume was the mean ± 25 µL.

HDL Stability: HDL was collected from the bottom fraction after flotation of TLP at d = 1.063 g/mL. Guanidinium chloride (GdmCl) (MP Biomedicals, LLC) and HDL were combined to produce final concentrations of 2 M and 0.5 mg/mL respectively. After three days at room temperature, the samples were analyzed by SEC and the relative amounts of lipid-free apo A-I and fused HDL determined.5 The effects of freezing on HDL were similarly assessed.

Hepatic HDL-CE Uptake: Cellular HDL-CE uptake was studied in the human hepatoma cell line, Huh7, which express the major lipoprotein receptors— scavenger receptor class B Type I (SR-BI), the low density lipoprotein receptor family members LDLR, LRP-1 and VLDLR, and heparan sulfate proteoglycan syndecan-1 (SDC1) (Supplemental Figure I). Cholesterol ester uptake was determined as described.6, 7 HDL functionality was based on a competitive assay of the inhibition of the cellular uptake of a standard HDL-[3H]CE by patient HDL. Huh7 cells were grown to confluence in 12-well plates, washed and transferred to DMEM + 0.5% fatty acid-free BSA. CE uptake assay was initiated by incubation of HDL-[3H]CE (20 µg HDL protein/mL) with

http://www.clinicaltrials.gov/

2

patient HDL in triplicate at 0, 20, 50 and 120 µg HDL protein/mL for 3 hr after which cells were washed, extracted with 2-propanol and the extract assayed for 3H-CE by β-counting. The data were fitted to a hyperbolic decay equation, i.e., %CE Uptake = aID50%/(ID50% + [I]), where [I] is the concentration of the added inhibitor protein, a is the y-intercept, and ID50% is the added inhibitor concentration that produces 50% inhibition.

Statistical Analysis. Statistical analysis and curve fitting were preformed in SigmaPlot 10 or 11. Multiple groups were compared using Kruskal-Wallis One Way Analysis of Variance on Ranks. Paired samples before and after treatment were compared using the Wilcoxon signed rank sum test. Other data were analyzed using the rank sum test, or when data were normally distributed, the Student t-test, as indicated in legends to Tables and Figures. References for Materials and Methods 1. Balasubramanyam A, Coraza I, Smith EO, Scott LW, Patel P, Iyer D, Taylor AA,

Giordano TP, Sekhar RV, Clark P, Cuevas-Sanchez E, Kamble S, Ballantyne CM, Pownall HJ. Combination of niacin and fenofibrate with lifestyle changes improves dyslipidemia and hypoadiponectinemia in HIV patients on antiretroviral therapy: Results of "Heart Positive," a randomized, controlled trial. J Clin Endocrinol Metab. 2011;96:2236-2247.

2. Samson SL, Pownall HJ, Scott LW, Ballantyne CM, Smith EO, Sekhar RV, Balasubramanyam A. Heart positive: Design of a randomized controlled clinical trial of intensive lifestyle intervention, niacin and fenofibrate for HIV lipodystrophy/dyslipidemia. Contemp Clin Trials. 2006;27:518-530.

3. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.

4. Pownall HJ, Brauchi D, Kilinc C, Osmundsen K, Pao Q, Payton-Ross C, Gotto AM, Jr., Ballantyne CM. Correlation of serum triglyceride and its reduction by omega-3 fatty acids with lipid transfer activity and the neutral lipid compositions of high-density and low-density lipoproteins. Atherosclerosis. 1999;143:285-297.

5. Pownall HJ, Hosken BD, Gillard BK, Higgins CL, Lin HY, Massey JB. Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning. Biochemistry. 2007;46:7449-7459.

6. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996;271:518-520.

7. Gillard BK, Rosales C, Pillai BK, Lin HY, Courtney HS, Pownall HJ. Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol. Biochemistry. 2010;49:9866-9873.

Table I: Heart Positive Plasma Lipid Values at Baseline and after 24 weeks of Treatment a Plasma Lipid

All HP patients

Treatment Groupb

1. Placebo 2. D&E 3. D&E+fibrate 4. D&E+niacin 5. D&E+fibrate+ niacin n=114 n=23 n=23 n=28 n=17 n=23 Baseline Treated pc Baseline Treated pc Baseline Treated pc Baseline Treated pc Baseline Treated pc

Cholesterol 195 205 197 0.218 184 198 0.474 202 181 0.010 184 182 0.243 201 179 0.064 mg/dL (172-230) (181-235) (178-225) (161-223) (181-220) (169-238) (156-222) (174-231) (158-210) (163-223) (146-219) Triglyceride 248 247 175 0.362 243 222 0.637 251 150 <0.001 282 165 0.023 203 131 <0.001 mg/dL (171-329) (182-321) (141-377) (157-312) (125-309) (195-340) (116-217) (201-424) (93-334) (158-300) (80-161) HDL-C 37 38 35 0.375 37 40 0.199 38 38 0.056 33 41 0.009 38 46 <0.001 mg/dL (31-42) (33-46) (30-50) (31-42) (33-49) (33-44) (34-49) (28-38) (31-44) (31-43) (40-55) Calc LDL-C 108 113 117 0.205 99 124 0.242 102 102 0.933 92 107 0.404 114 107 1.000 mg/dL (77-137) (81-146) (77-146) (84-123) (100-133) (73-146) (86-125) (60-119) (79-127) (80-140) (84-127)

Non-HDL-C 158 175 159 0.266 153 158 0.903 163 130 0.002 149 141 0.130 170 127 0.006 mg/dL (135-187) (141-192) (128-189) (125-181) (145-178) (132-201) (109-171) (142-196) (120-172) (127-181) (105-172)

a. Plasma lipid values are given as median and (25%-75%) range. Normal values are Cholesterol <200 mg/dL; Triglycerides <150

mg/dL; HDL-C > 45 – 55 mg/dL; Calculated LDL-C <130 mg/dL and Non-HDL-C <160 mg/dL. b. Heart Postive patients were randomized to 5 groups. Kruskal-Wallis One Way Analysis of Variance on Ranks of the lipid values

for the five groups at baseline confirmed that the lipid values at baseline did not differ among the groups ( p values of 0.088 to 0.812).

c. Wilcoxon Signed Rank Test comparing paired values at baseline and after treatment.

Supplemental Figure I: A, Phase contrast image for Huh7 cells; note lipid droplets and confluent cobblestone monolayer architecture compared to HepG2 cells which tend to grow in overlaying clusters. B, mRNA expression for major lipoprotein receptors in Huh7 cells compared to expression in HepG2 cells and primary human hepatocytes at days 2 and 5 of culture. Expression levels of the low density lipoprotein receptor family members LDLR, LRP1, and VLDLR, Scavenger Receptor Class B Type I SR-BI (SCARB1) and heparan sulfate proteoglycan Syndecan-1 (SDC1) were determined by quantitative reverse transcription PCR (qRTPCR) and normalized to 18S for each cell type.

0

100

200

300

400

LDLR LRP1 SCARB1 SDC1 VLDLR

mR

NA,

18S

Nor

mal

ized

Dat

a

B. Hepatocyte Lipoprotein Receptor mRNA levels

Huh7

HepG2

Hu8089Day2

Hu8089Day5

A. Huh7 cells, phase contrast

Smallest HDLSmallest LDL

Largest LDL

B. NL Control TLP Chromatograms

Smallest LDL

Largest LDL

A. HIV TLP Chromatograms

Smallest HDLLargest LDL17NL

18NL

21NL

23NL

Smallest HDLSmallest LDL

20HIV

19HIV

16HIV 23NL

7NL

6NL

16HIV

13HIV

14HIV

4NL

3NL

8NL

15HIV

1HIV

12HIV

9NL

2NL

12HIV

11HIV

10HIV

Elution Volume, mL

15 20 25 30 35

5NL

Elution Volume, mL

15 20 25 30 35

Supplemental Figure II. Comparison of Lipoprotein Sizes bySEC of TLP from HIV/ART and NL Control Subjects. Tenindividual donor HIV/ART TLP chromatograms are shown in PanelA, and 12 individual donor NL Control TLP chromatograms areshown in Panel B. These chromatograms were used to calculatethe mean and SEM profiles shown in Figure 4.

0.20.40.60.81.01.2

b = 71.8 + 3.5r2 >0.99

0.20.40.60.81.01.2 C-1HIV

B-7NL

HDL-Inhibitor, g/mL0 25 50 75 100 125

0.20.40.60.81.01.2

D-11HIV

A-17NLb = 86.9 + 4.2r2 >0.99

b = 71. + 9.0r2 >0.98

b = 80.1 + 21.1r2 >0.93

b = 114.9 + 11.8r2 >0.99

b = 94.1 + 7.1r2 >0.96

b = 94.1 + 7.1r2 >0.99

b = 82.5 + 16.7r2 >0.96

CE

Upt

ake,

%C

ontr

ol

0.20.40.60.81.01.2

impaired lipoprotein processing in hiv patients on...

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