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, 36 (2), 274-84

Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties

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Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties

Su Duy Nguyen et al. Arterioscler Thromb Vasc Biol.

Abstract

Objective: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I.

Approach and results: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils.

Conclusions: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

Keywords: apolipoprotein A-I; carboxyl-terminal cleavage; chymase; endothelial cells; inflammatory; mast cell; proteases.

Figures

Figure 1.
Figure 1.
Chymase treatment abolishes the anti-inflammatory activity of apolipoprotein A-I (apoA-I) on activated human coronary artery endothelial cells (HCAECs). A, HCAECs were preincubated for 16 h in the absence (buffer) or presence of apoA-I (50 μg/mL) and then activated with tumor necrosis factor-α (TNF-α; 10 ng/mL) for 5 h. Nonactivated cells were incubated in medium alone for 21 h (control). Cell surface vascular cell adhesion molecule-1 (VCAM-1) protein was determined by flow cytometry. Data are representative of 6 independent experiments. B, ApoA-I (1 mg/mL) was treated for 6 h in the absence (untreated) or the presence (chymase-treated) of chymase (0.5 μg=40 BTEE units/mL). HCAECs were preincubated with increasing concentrations of the untreated or chymase-treated apoA-I and then activated with tumor necrosis factor-α (TNF-α), as described in A. TNF-α-induced VCAM-1 surface protein expression was analyzed by flow cytometry and expressed as percentage of its expression levels in TNF-α-activated cells preincubated in the absence of apoA-I, which was set as 100%. Data represent the means±SD from 3 to 4 independent experiments performed in duplicate. *P<0.01 denotes statistical significance between cells preincubated with untreated or chymase-treated apoA-I. C, ApoA-I was incubated with the indicated activities (BTEE units) of recombinant human chymase or chymase-containing human mast cell–conditioned medium for 6 h, after which the incubation was stopped by adding soybean trypsin inhibitor. Proteins in the incubation mixtures were resolved in 12.5% SDS-PAGE and detected by Coomassie Blue or immunoblotted with anti-human apoA-I polyclonal antibody or with anti-human apoA-I monoclonal antibodies recognizing either a C-terminal (amino acids 211–220) or an N-terminal (amino acids 2–8) region of apoA-I. MFI indicates median fluorescence intensity.
Figure 2.
Figure 2.
Effect of apolipoprotein A-I (apoA-I) lipidation on its sensitivity to proteolysis and on the ability of untreated and chymase-treated apoA-I to influence vascular cell adhesion molecule-1 (VCAM-1) expression in and THP-1 adhesion to tumor necrosis factor-α (TNF-α)-activated human coronary artery endothelial cells (HCAECs). A, Top, ApoA-I and discoidal apoA-I-containing reconstituted high-density lipoprotein ((A-I)rHDLs) with 3 different degrees of lipidation (see Table I in the online-only Data Supplement) were incubated in the absence or presence of recombinant chymase, and the proteins in the incubation mixtures were analyzed, as described in Figure 1. Bottom, ApoA-I degradation was expressed as a percentage of the intensity of the intact band in the proteolyzed samples relative to the corresponding untreated sample and plotted against the lipid/protein mass ratio of the various (A-I)rHDL preparations. B, HCAECs were preincubated for 16 h with the various untreated or chymase-treated apoA-I or (A-I)rHDL species (50 μg/mL, each) and then activated with tumor necrosis factor-α (TNF-α), as described in Figure 1. Nonactivated cells (control) and TNF-α-activated cells preincubated in only medium (buffer) acted as references. Vascular cell adhesion molecule-1 (VCAM-1) mRNA levels (top, fold change relative to control) and surface protein expression (middle, % of buffer) were evaluated in the TNF-α-activated cells. In a separate experiment, HCAECs that had been preincubated with the various apoA-I-containing preparations were further incubated for 1 h with fluorescently labeled THP-1cells, and the fluorescence of HCAECs-bound THP-1 cells was then measured (bottom). The fluorescence signal of the TNF-α-activated HCAECs was expressed as percentage of the basal signal from the nonactivated cells (control) which was set as 100%. Data shown in each panel represent the means±SD from 3 to 4 independent experiments performed in triplicate wells. **P<0.05; *P<0.01; #P<0.01 (untreated apoA-I-containing preparations vs buffer).
Figure 3.
Figure 3.
Proteolysis of apolipoprotein A-I (apoA-I) impairs its ability to attenuate nuclear factor-κB (NF-κB) activation to bind to human coronary artery endothelial cells (HCAECs) and to prevent transmigration of THP-1 monocytes across HCAEC monolayer. A, HCAECs were incubated for 16 h with untreated or chymase-treated apoA-I and then activated with tumor necrosis factor-α (TNF-α) for 15 min. Nonactivated cells (control) and TNF-α-activated cells preincubated in only medium (buffer) acted as a reference. Nuclear NF-κB p65 activity was determined by measuring nuclear translocation of the NF-κB p65 subunit. The basal activity of the nonactivated HCAECs (control) was set as 1. Data represent the means±SD from triplicate wells and are representative of 2 independent experiments. *P<0.05. B, ApoA-I was labeled with 125I and then treated with chymase as described in Figure 1B. HCAECs were incubated with the indicated concentrations of 125I-labeled untreated or chymase-treated apoA-I in the absence or presence of a 40-fold excess of untreated apoA-I for 2 h at 4°C. High-affinity binding of 125I-apoA-I to HCAECs was calculated by subtracting the values of the nonspecific binding from the total binding. A representative pair of binding curves is shown in the top panel. Data (mean±SD) from 4 independent experiments from duplicate wells are expressed as percentages of untreated apoA-I (bottom). *P<0.01. C, Confluent HCAEC monolayers grown on a transwell insert were first incubated with untreated or chymase-treated apoA-I added either to the apical or basolateral compartment, then activated with TNF-α for 5 h, and finally incubated with fluorescently labeled THP-1 cells added to the apical compartment. Migration across the endothelial monolayer of the cells was quantified by measuring the fluorescence signal of the transmigrated cells. Migration in the absence of any additions was designated as 100% (control). Data shown represent the means±SD from 3 independent experiments performed in triplicate wells. *P<0.05; #P<0.05 (untreated apoA-I vs buffer).
Figure 4.
Figure 4.
Proteolysis of apolipoprotein A-I (apoA-I) reduces its ability to downregulate the expression of proinflammatory genes in human coronary artery endothelial cells (HCAECs) and in human GM-Mac and M-Mac macrophage subpopulations and to promote cholesterol efflux from the macrophages. A-D, HCAECs were preincubated with untreated or chymase-treated apoA-I and then activated with tumor necrosis factor-α (TNF-α), as described in Figure 1. Nonactivated cells (control) and TNF-α-activated cells preincubated in only medium (buffer) acted as a reference. The mRNA levels of intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and IL-8 in HCAECs were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and expressed as fold changes relative to the control cells. Data represent the means±SD from 3 independent experiments performed in duplicate. *P<0.01. E-L, Human monocyte-derived macrophages were differentiated in the presence of GM-CSF (granulocyte-macrophage colony-stimulating factor) or M-CSF (macrophage colony-stimulating factor) into GM-Mac and M-Mac subtypes, respectively, and then converted into foam cells by incubation with acetyl low density lipoprotein (LDL). The cells were then incubated for 3 h with untreated or chymase-treated apoA-I (50 μg/mL, each), washed, and activated for 3 h with lipopolysaccharide (LPS). Nonactivated cells (control) and LPS-activated cells preincubated in only medium (buffer) acted as references. LPS-induced mRNA levels of TNF-α, IL-1β, IL-6, and IL-8 in GM-Mac and M-Mac foam cells were evaluated by qRT-PCR and expressed as fold changes relative to the control cells. *P<0.01; **P<0.05. M, GM-Mac- and M-Mac-derived foam cells were incubated with untreated or chymase-treated apoA-I (50 μg/mL, each) for 3 h, the media were collected, centrifuged to remove cellular debris, and the radioactivity of each supernatant was determined by liquid scintillation counting. Cells were solubilized, and radioactivity was determined in the cell lysates. Cholesterol efflux was calculated as dpmmedium/(dpmcells+dpmmedium)×100. Data represent the means±SD from triplicate wells and are representative of 2 independent experiments. *P<0.01.
Figure 5.
Figure 5.
Proteolysis of apolipoprotein A-I (apoA-I) impairs its ability to inhibit lipopolysaccharide (LPS)-induced inflammation in vivo and to neutralize endotoxin activity of LPS in vitro. Mice (6–8 per group) were randomized to receive vehicle (PBS), LPS (1 mg/kg), LPS (1 mg/kg) plus apoA-I (10 mg/kg), or LPS (1 mg/kg) plus chymase-treated apoA-I (10 mg/kg). After 3 h, serum was collected, and TNF-α (A) and IL-1β (B) concentrations were measured. Data are means±SD, *P<0.05. C, LPS (1 μg/mL) was mixed with untreated apoA-I and chymase-treated apoA-I (10 μg/mL) and endotoxin activity was measured with kinetic colorimetric limulus amebocyte lysate (LAL) assay. Data represent the means±SD from triplicate wells and are representative of 2 independent experiments.
Figure 6.
Figure 6.
Chymase treatment does not affect the anti-inflammatory effects of D-4F on human coronary artery endothelial cells (HCAECs), neutrophils, and macrophages. A, HCAECs were preincubated with untreated or chymase-treated L-4F and D-4F (50 μg/mL, each) after which the cells were then activated with tumor necrosis factor-α (TNF-α), as described in Figure 1. Nonactivated cells (control) and TNF-α-activated cells preincubated in only medium (buffer) were used as references. Cell-surface expression of vascular cell adhesion molecule-1 (VCAM-1) protein was determined by flow cytometry. Data represent the means±SD from 3 independent experiments performed in duplicate. *P<0.01; #P<0.01 (untreated L-4F or untreated D-4F vs buffer). B, Superoxide radical production by PMA (phorbol 12-myristate 13-acetate)–activated neutrophils was determined after the cells had been preincubated for 5 min with the indicated concentrations of untreated or chymase-treated L-4F, D-4F, or apolipoprotein A-I (apoA-I). PMA-activated neutrophils preincubated in only medium (buffer) were used as a reference (open triangles). Luminescence emitted by nonactivated neutrophils (control) was set as 1. Data are derived from 2 donors and represent the means±SD from 2 independent experiments, each performed in 3 to 6 wells. *P<0.01 (untreated vs chymase-treated L-4F, and untreated vs chymase-treated apoA-I); #P<0.01 (untreated L-4F, D-4F, or apoA-I vs buffer). C-J, Human monocyte–derived macrophages were differentiated into GM-Mac and M-Mac subtypes and converted into foam cells by incubation with acetyl low-density lipoprotein (LDL). The foam cells were then incubated for 3 h with untreated or chymase-treated D-4F (50 μg/mL, each), washed, and activated for 3 h with lipopolysaccharide (LPS). Nonactivated foam cells (control) and LPS-activated foam cells preincubated in only medium (buffer) served as references. LPS-induced mRNA levels of TNF-α, interleukin (IL)-1β, IL-6, and IL-8 in GM-Mac and M-Mac foam cells were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and expressed as fold changes relative to the control cells. Data represent the means±SD from triplicate wells and are representative of 2 independent experiments. *P<0.01; **P<0.05.

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