Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.

Apolipoproteins are lipoprotein (LP)-associated proteins that stabilize LP structure and mediate receptor-dependent LP recognition. LP’s interaction with receptors, such as the low-density LP (LDL) receptor or class B scavenger receptors (SR-Bs; SR-BI, SR-BII, or CD36), controls many aspects of lipid metabolism. In addition, SR-Bs mediate pathogen recognition and innate and adaptive immune responses (13). Apolipoprotein A-I (apoA-I) is the major protein of high-density LP (HDL), which plays an important role in reverse cholesterol transport, as well as possesses anti-inflammatory and tissue-protecting properties (47). Mice overexpressing the human apoA-I gene have increased levels of HDL and reduced chronic inflammation and are less prone to atherosclerosis. In addition, administration of HDL to cholesterol-fed rabbits induced the regression of atherosclerotic lesions (5). apoA-I attenuates atherosclerosis via reverse cholesterol transport from macrophages residing in atherosclerotic plaques. Subsequently, cholesterol is transported to the liver and excreted into the gastrointestinal tract (6). apoA-I also reduces inflammation triggered by reactive oxygen species (8), oxidized LPs (9), various proinflammatory bacteria-derived products (e.g., LPS and chaperonin 60), bacteria (10, 11), and acute-phase proteins, such as serum amyloid A (12, 13). These processes are mediated, in part, by apoA-I blocking various receptors sensing danger-associated molecular patterns (DAMPs), including proinflammatory signals (14, 15). It was demonstrated that synthetic amphipathic helical peptides (SAHPs), also known as apoA-I mimetic peptides (because of their ability to mediate some of apoA-I’s functions), antagonize various apoA-I binding receptors, such as formyl peptide receptors, lectin-like oxidized low-density LP receptor (LOX)-1, and class A and class B scavenger receptors, including SR-BI, its splicing variant SR-BII, and CD36 (1618). SR-Bs are highly expressed in phagocytes, lung epithelial cells, liver hepatocytes, and endothelial cells (ECs), indicating that SR-B may be of critical importance in the development of acute pulmonary syndromes (3, 6, 1921). In this respect, SAHPs and HDLs that have the ability to antagonize CD36/SR-BI may have beneficial effects on EC functions, including control of the EC barrier and antithrombotic activity (9). Indeed, SAHPs exhibit multiple anti-inflammatory properties (6, 9, 22) and interrupt proinflammatory signal-transduction cascades, such as sphingosine kinase– and ERK-mediated signaling pathways, as well as NF-κB activation induced by ligands of SR-BI/II and CD36 (23). SAHPs also attenuate the generation of reactive oxygen species, inhibit proteasome activation, and reduce matrix metalloproteinase expression (1, 8).

Acute inflammation–related syndromes, such as bacteria/LPS-induced systemic shock, sepsis, blood loss, crash syndromes, acute lung injury (ALI), and pulmonary edema, are associated with endothelial barrier dysfunction and represent life-threatening syndromes associated with high morbidity and mortality. It is established that the TLR system makes the major contribution to inflammatory responses in many of these cases (24), including ALI (25). However, recent data demonstrate that SR-Bs, especially CD36, may significantly facilitate inflammatory responses during endotoxemia (26, 27) and sepsis (28). We propose that these two receptor systems act in a complementary manner in the pulmonary response to pathogen associated molecular patterns. However, there is an additional effect of CD36 due to LPS-induced cell injury that potentially results in the release of cell-generated DAMPs, which are known to exert their inflammatory effects via CD36 (6, 7). Because current options for ALI treatment are limited, new approaches, especially those based on CD36 targeting, are warranted.

Based upon the anti-inflammatory effects of apoA-I and SAHPs, there has been considerable interest in developing novel SAHP-based therapies as a way to decrease and control such serious inflammatory conditions. One approach to apoA-I–based therapies has been the development of anti-inflammatory apoA-I mimetic peptides (SAHPs) targeting apoA-I receptors, such as SR-BI and CD36, as well as other receptors (4, 6, 29). apoA-I mimetic peptides have an amphipathic α helical structure that is similar to the native apoA-I protein secondary structure that contains 10 such amphipathic α helices (29). Minimal changes in the apoA-I mimetic peptide sequence affecting polar and nonpolar interfaces oriented along the long axis of the peptide helix region may change specific interactions with various receptors, including SR-BI/II and CD36 (30). Based on this approach, we tested a group of SAHP analogs having minimal sequence changes to select the best apoA-I mimetics, specifically targeting CD36 versus SR-BI/II, and designed to treat acute inflammation syndromes, such as LPS-induced ALI.

Twenty-four apoA-I mimetic peptides were synthesized. Their sequences, physicochemical properties, and general features are summarized in Table I. Two peptides were used as prototypes to understand how structural modifications affect their function. The first prototype peptide, 5A (Table I, no. 22), was described previously by our group (31). This peptide consists of two type A amphipathic α helices connected by a proline. The hydrophobicity of the second helix was reduced by substitution of hydrophobic amino acids with alanine. Two derivatives of 5A were synthesized (peptides 23 and 24) to test the impact of the introduction of 2 aa with antioxidant potential: cysteine and histidine. The second prototype peptide, ELK (Table I, no. 1), contains only 3 aa residues: glutamic acid, leucine, and lysine. It consists of two identical canonical type A amphipathic α helices with the hydrophobic interface turned by 180° and neutral net charge. The helices within this peptide are connected by a proline residue. The original ELK peptide was used to make 19 derivatives (Table I, peptides 2–20). Several parameters, in particular net charge, mean hydrophobicity and the hydrophobic face size, type of helix, and configuration of the proline bridge between the two helices, which may affect the peptide interaction with LPs and cellular receptors, as well as peptide structural asymmetry to affect cholesterol efflux specificity (31), were modified. Because some of these properties (e.g., charge and hydrophobicity) are interdependent, additional testing of several peptides with combinations of these features was performed.

Table I.
Amino acid sequences of SAHPs tested in this study
NameFormula for ELK-Based Peptides
ELK EKLKELLEKLLEKLKELLPEKLKELLEKLLEKLKELL 
ELK-B EKLLELLKKLLELLKKLLPEKLLELLKKLLELLKKLL 
ELK-B2 EKLKELLEKLLELLKKLLPEKLKELLEKLLELLKKLL 
ELK-C EELKEKLEELKEKLEEKLPEELKEKLEELKEKLEEKL 
ELK-C1 EELKAKLEELKAKLEEKLPEELKAKLEELKAKLEEKL 
ELK-C3 EKLKELLEKLKAKLEELLPEKLKELLEKLKAKLEELL 
ELK-C4 EKLKAKLEELKAKLEELLPEKLKAKLEELKAKLEELL 
ELK-D EKLKALLEKLLAKLKELLPEKLKALLEKLLAKLKELL 
ELK-D2 EKLKELLEKLLAKLKELLPEKLKELLEKLLAKLKELL 
ELK-E EWLKELLEKLLEKLKELLPEWLKELLEKLLEKLKELL 
ELK-F EKFKELLEKFLEKFKELLPEKFKELLEKFLEKFKELL 
ELK-F2 EKFKELLEKLLEKLKELLPEKFKELLEKLLEKLKELL 
ELK-G EELKELLKELLKKLEKLLPEELKELLKELLKKLEKLL 
ELK-H EELKKLLEELLKKLKELLPEELKKLLEELLKKLKELL 
ELK-I EKLKELLEKLLEKLKELLAEKLKELLEKLLEKLKELL 
ELK-J EKLKELLEKLLEKLKELLAAEKLKELLEKLLEKLKELL 
ELK-K DWLKAFYDKVACKLKEAFPDWAKAAYNKAAEKAKEAA 
ELK-L DHLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 
ELK-A2 EKLKAKLEELKAKLEELLPEKAKAALEEAKAKAEELA 
ELK-AS EKLKAKLEELKAKLEELLPEHAKAALEEAKCKAEELA 
L37PA DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 
5A DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 
P5A DHLKAFYDKVACKLKEAFPNWAKAAYDKAAEKAKEAA 
P5A C12/H2 DWLKAFYDKVAEKLKEAFPDHAKAAYDKAACKAKEAA 
NameFormula for ELK-Based Peptides
ELK EKLKELLEKLLEKLKELLPEKLKELLEKLLEKLKELL 
ELK-B EKLLELLKKLLELLKKLLPEKLLELLKKLLELLKKLL 
ELK-B2 EKLKELLEKLLELLKKLLPEKLKELLEKLLELLKKLL 
ELK-C EELKEKLEELKEKLEEKLPEELKEKLEELKEKLEEKL 
ELK-C1 EELKAKLEELKAKLEEKLPEELKAKLEELKAKLEEKL 
ELK-C3 EKLKELLEKLKAKLEELLPEKLKELLEKLKAKLEELL 
ELK-C4 EKLKAKLEELKAKLEELLPEKLKAKLEELKAKLEELL 
ELK-D EKLKALLEKLLAKLKELLPEKLKALLEKLLAKLKELL 
ELK-D2 EKLKELLEKLLAKLKELLPEKLKELLEKLLAKLKELL 
ELK-E EWLKELLEKLLEKLKELLPEWLKELLEKLLEKLKELL 
ELK-F EKFKELLEKFLEKFKELLPEKFKELLEKFLEKFKELL 
ELK-F2 EKFKELLEKLLEKLKELLPEKFKELLEKLLEKLKELL 
ELK-G EELKELLKELLKKLEKLLPEELKELLKELLKKLEKLL 
ELK-H EELKKLLEELLKKLKELLPEELKKLLEELLKKLKELL 
ELK-I EKLKELLEKLLEKLKELLAEKLKELLEKLLEKLKELL 
ELK-J EKLKELLEKLLEKLKELLAAEKLKELLEKLLEKLKELL 
ELK-K DWLKAFYDKVACKLKEAFPDWAKAAYNKAAEKAKEAA 
ELK-L DHLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 
ELK-A2 EKLKAKLEELKAKLEELLPEKAKAALEEAKAKAEELA 
ELK-AS EKLKAKLEELKAKLEELLPEHAKAALEEAKCKAEELA 
L37PA DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 
5A DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 
P5A DHLKAFYDKVACKLKEAFPNWAKAAYDKAAEKAKEAA 
P5A C12/H2 DWLKAFYDKVAEKLKEAFPDHAKAAYDKAACKAKEAA 

All cell-uptake studies were performed using DMEM containing 2 mg/ml BSA, as described elsewhere. HeLa cells stably transfected with SR-BI, SR-BII, and CD36 were incubated with Alexa Fluor 488–labeled HDL, Alexa Fluor 488–labeled LDL, or Alexa Fluor 488–labeled L37pA (10 μg/ml, 37°C, for 1 h), washed with PBS, and detached with Cellstripper dissociation solution (Cellgro, Herndon, VA). The detached cells were fixed with 4% paraformaldehyde and analyzed by a fluorescence-activated cell sorter (model A) or using a Victor3 fluorometer (Perkin Elmer).

Measurement of transendothelial electrical resistance (TER) across confluent human pulmonary artery EC monolayers was performed with an electrical cell substrate impedance-sensing system (Applied Biophysics, Troy, NY), as previously described (32). Cell monolayers were challenged with LPS (200 ng/ml), with or without pretreatment with SAHPs, and TER measurements were performed over 20 h.

Endothelial and THP-1 cells were cultured as described elsewhere. HEK293 cells stably transfected to express human SR-BI, human SR-BII, and human CD36 were reported previously (33). The cells were grown to 50% confluency in DMEM containing 10% FBS and antibiotics. After a 24-h incubation in serum-free DMEM, cells were challenged with various concentrations of Salmonella minnesota LPS in the presence or absence of various peptides (10 μg/ml) for an additional 24 h. Collected culture medium was analyzed for IL-8 secretion using ELISA (11, 26, 34). Peptides demonstrating selective inhibition of LPS-induced IL-8 secretion through the CD36 pathway versus the SR-BI/II pathway were selected and used for in vivo experiments.

All animal care and treatment procedures were approved by the Animal Care and Use Committees of the National Heart, Lung, and Blood Institute and the University of Chicago. Animals were handled according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. C57BL/J male mice (8–10-wk old), with an average weight of 20–25 g, were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were anesthetized by i.p. injection of ketamine (75 mg/kg) and acepromazine (1.5 mg/kg). Bacterial LPS (0.63 mg/kg body weight; Escherichia coli O55:B5) or sterile water was injected intratracheally in a small volume (20–30 μl) using a 20-gauge catheter. SAHPs were injected i.v. 30 min after LPS intratracheal instillation. Peptide and saline solutions were essentially LPS free, as tested by Limulus amebocyte lysate. After 16 h, animals were sacrificed by exsanguination under anesthesia. Bronchoalveolar lavage was performed using 1 ml sterile Hanks’ balanced salt buffer, and measurements of cell count, protein concentration, and myeloperoxidase activity were conducted as previously described (35).

Results are presented as mean ± SEM. One-way ANOVA with a Bonferroni multiple-comparison test or two-way ANOVA with a Bonferroni posttest test was calculated using GraphPad Prism, version 5.0a (GraphPad, La Jolla, CA). A p value < 0.05 was considered significant.

Previous studies demonstrated that SR-BI, SR-BII, and CD36 bind various LPs, including HDL and its apolipoproteins (apoA-I and apolipoprotein A-II) (36, 37). Because the specificity of SAHPs is not well understood, the uptake of L37pA and its inactive analog L3D-37pA was first evaluated in HeLa cell lines stably transfected with human SR-BI, SR-BII, and CD36 and compared with two canonical SR-B ligands: HDL and LDL. The uptake of Alexa Fluor 488–labeled HDL, Alexa Fluor 488–labeled LDL, and Alexa Fluor 488–labeled L37pA was significantly increased in human SR-BI and CD36 overexpressing HeLa cells compared with mock-transfected controls (Fig. 1A). No increase was found for L3D-37pA (data not shown). Furthermore, no increase in Alexa Fluor 488–labeled BSA or Alexa Fluor 488–labeled lactoferrin uptake was seen in these cells compared with mock-transfected HeLa controls (data not shown). Uptake of L37pA was dose dependent and demonstrated a similar dose response in HeLa cells overexpressing SR-BI, SR-BII, and CD36. Both mock-HeLa and LDL receptor overexpressing HeLa minimally bound Alexa Fluor 488–labeled L37pA (Fig. 1B).

FIGURE 1.

SR-B ligand uptake in human SR-BI–, SR-BII–, and CD36-overexpressing HeLa cells. (A) HeLa cells stably transfected with SR-BI, SR-BII, and CD36 were incubated without any ligands or with Alexa Fluor 488–labeled HDL, Alexa Fluor 488–labeled LDL, or Alexa Fluor 488–labeled L37pA for 2 h at 37°C, followed by FACS analysis. (B) Dose-dependent binding of Alexa Fluor 488–labeled ligands to cultured HeLa cell expressing various receptors.

FIGURE 1.

SR-B ligand uptake in human SR-BI–, SR-BII–, and CD36-overexpressing HeLa cells. (A) HeLa cells stably transfected with SR-BI, SR-BII, and CD36 were incubated without any ligands or with Alexa Fluor 488–labeled HDL, Alexa Fluor 488–labeled LDL, or Alexa Fluor 488–labeled L37pA for 2 h at 37°C, followed by FACS analysis. (B) Dose-dependent binding of Alexa Fluor 488–labeled ligands to cultured HeLa cell expressing various receptors.

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Lipid-free L37pA, as well as its complex with phosphatidylcholine, ameliorated LPS-induced IL-8 secretion by cultured human lung microvascular pulmonary ECs (HLMVECs) in a dose-dependent manner, whereas 3D-37pA peptide had no effect (Fig. 2). L37pA also attenuated LPS-induced IL-8 secretion in THP-1, fibroblasts, and A549 pulmonary epithelial cell lines (data not shown).

FIGURE 2.

Effect of L37pA and its phospholipidated form on LPS-induced IL-8 secretion in HLMVECs. After HLMVECs were grown to full confluency, cell monolayers were washed with PBS and incubated in serum-free medium for 24 h. LPS (10 ng/ml) in serum-free medium containing penicillin and streptomycin was added to HLMVECs, and conditioned medium was collected for IL-8 measurements 24 h later.

FIGURE 2.

Effect of L37pA and its phospholipidated form on LPS-induced IL-8 secretion in HLMVECs. After HLMVECs were grown to full confluency, cell monolayers were washed with PBS and incubated in serum-free medium for 24 h. LPS (10 ng/ml) in serum-free medium containing penicillin and streptomycin was added to HLMVECs, and conditioned medium was collected for IL-8 measurements 24 h later.

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Because L37pA binds to hSR-BI (33), as well as to human SR-BII and human CD36 receptors (Fig. 1), we further tested a panel of SAHPs based on a simple design using a combination of three amino acids (Table I): the hydrophilic positively charged lysine (K), negatively charged glutamic acid (E), and hydrophobic leucine (L), which are referred to as ELK peptides. This peptide panel was used to identify SAHPs selectively targeting CD36 rather than SR-BI/II. Using HEK293 cell lines stably transfected with human SR-BI, SR-BII, and CD36, we found that ELK-B and ELK-B2 blocked IL-8 secretion induced by LPS in cells expressing CD36 but were less effective in blocking LPS-induced IL-8 secretion in cells expressing SR-BI/II (Table II). The majority of tested peptides had either no effect or affected both receptors (L37pA and 5A), leading to a dose-dependent suppression of LPS-induced IL-8 secretion in all cell lines (Fig. 3). These data identify ELK-B and ELK-B2 peptides as the most efficient and relatively selective antagonists of LPS-induced IL-8 secretion in cells expressing the CD36 receptor.

Table II.
Effect of ELK-based SAHPs on LPS-induced IL-8 secretion using HEK293 cells stably transfected with human SR-BI, human SR-BII, or human CD36
 
 

Cells were incubated with 10 ng/ml LPS in the presence or absence of various SAHPs for 24 h in serum-free DMEM. Conditioned media were analyzed for IL-8 secretion using ELISA.

Predominant SR-BI/II or CD36 targeting or similar CD36–SR-BI/II targeting are highlighted in blue, yellow, and green, respectively. Noneffective peptides are highlighted in red.

FIGURE 3.

Effects of SAHPs on LPS-induced IL-8 secretion in HEK293 cells. (A) WT and human CD36 stably transfected HEK293 cells were treated with LPS (0, 20, 50, 100, 250, or 500 ng/ml) for 24 h. IL-8 was measured as pg/mg of cell protein. (B) CD36-expressing cells were incubated with 50 ng/ml of LPS in the presence or absence of increasing concentrations of various SAHPs for 24 h in serum-free media. IL-8 was measured as a percentage of inhibition in human CD36 stably transfected HEK293 cells.

FIGURE 3.

Effects of SAHPs on LPS-induced IL-8 secretion in HEK293 cells. (A) WT and human CD36 stably transfected HEK293 cells were treated with LPS (0, 20, 50, 100, 250, or 500 ng/ml) for 24 h. IL-8 was measured as pg/mg of cell protein. (B) CD36-expressing cells were incubated with 50 ng/ml of LPS in the presence or absence of increasing concentrations of various SAHPs for 24 h in serum-free media. IL-8 was measured as a percentage of inhibition in human CD36 stably transfected HEK293 cells.

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We further evaluated the effects of SAHPs in the models of human lung microvascular EC barrier dysfunction induced by LPS. EC monolayers were treated with LPS, with or without SAHP pretreatment, followed by measurement of transendothelial permeability. LPS increased HLMVEC monolayer permeability, which was significantly attenuated by L37pA pretreatment, whereas the L3D-37pA peptide was without effect (Fig. 4).

FIGURE 4.

Effect of L37pA and L3D-37pA on endothelial barrier function in vitro. Human microvascular lung ECs were grown on gold microelectrodes and used for TER measurements, as described in 2Materials and Methods. Cells were pretreated with L37pA (A) or its inactive homolog, L3D-37pA (B), at two concentrations (10 and 100 μg/ml), followed by LPS (200 ng/ml) challenge at the times indicated by arrows. Control cells were treated with LPS or vehicle. L37pA (100 μg/ml) abolished the LPS-induced endothelial barrier disruption reflected by TER decline.

FIGURE 4.

Effect of L37pA and L3D-37pA on endothelial barrier function in vitro. Human microvascular lung ECs were grown on gold microelectrodes and used for TER measurements, as described in 2Materials and Methods. Cells were pretreated with L37pA (A) or its inactive homolog, L3D-37pA (B), at two concentrations (10 and 100 μg/ml), followed by LPS (200 ng/ml) challenge at the times indicated by arrows. Control cells were treated with LPS or vehicle. L37pA (100 μg/ml) abolished the LPS-induced endothelial barrier disruption reflected by TER decline.

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The anti-inflammatory effects of SAHPs were further tested in a murine model of LPS-induced lung injury. LPS was injected intratracheally, with or without concurrent i.v. administration of peptides. Control mice were treated with vehicle (saline solution) or L3D-37pA. At the end of the experiment, lung injury was evaluated by measurement of total cell count, protein concentration, and concentrations of PMN cells in bronchoalveolar lavage fluid (BALF). An LPS challenge caused pronounced lung inflammation that was characterized by a dramatic increase in total cell and neutrophil counts in BALF, as well as an increase in BALF protein content. A single i.v. injection of L37pA at the beginning of the experiment significantly attenuated LPS-induced neutrophil and protein accumulation in BALF (Fig. 5A–C). Mouse treatment with ELK-B, which more specifically targets CD36, caused a more pronounced inhibitory effect on LPS-induced protein accumulation and cell count increase in BALF (Fig. 5D–F). In contrast, ELK predominantly targeting SR-BI and SR-BII demonstrated minimal effects on these parameters of LPS-induced lung damage (Figs. 5, 6).

FIGURE 5.

Effect of L37pA, L3D-37pA, 5A, ELK, and ELK-B on LPS-induced lung barrier dysfunction and neutrophil accumulation in BALF. L37pA and its inactive analog, L3D-37pA, were injected at the indicated doses into the jugular vein concurrently with intratracheal administration of LPS, as described in 2Materials and Methods. Total cell count (A), neutrophil count (B), and protein concentration (C) in BALF samples. ELK, ELK-B, and 5A peptides were injected at the indicated doses into the jugular vein concurrently with intratracheal LPS administration, as described in 2Materials and Methods. Total cell count (D), neutrophil count (E), and protein concentration (F) in BALF samples. Data are mean ± SE (n = 6/group).

FIGURE 5.

Effect of L37pA, L3D-37pA, 5A, ELK, and ELK-B on LPS-induced lung barrier dysfunction and neutrophil accumulation in BALF. L37pA and its inactive analog, L3D-37pA, were injected at the indicated doses into the jugular vein concurrently with intratracheal administration of LPS, as described in 2Materials and Methods. Total cell count (A), neutrophil count (B), and protein concentration (C) in BALF samples. ELK, ELK-B, and 5A peptides were injected at the indicated doses into the jugular vein concurrently with intratracheal LPS administration, as described in 2Materials and Methods. Total cell count (D), neutrophil count (E), and protein concentration (F) in BALF samples. Data are mean ± SE (n = 6/group).

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FIGURE 6.

Comparative structure analysis of peptides exhibiting anti-inflammatory properties and inactive peptides. L37pA has two identical amphipathic helices, called 18A, which are connected with a proline (hydrophobic residues are shown in yellow). The 5A peptide also has two amphipathic helices connected with a proline: 18A and modified 18A, where 5 aa are substituted with alanine, to make this second helix more hydrophobic. Two identical helices of the ELK peptide consist of three types of residues: hydrophobic leucine, positively charged lysine, and negatively charged glutamic acid. Helices have hydrophobic and hydrophilic phases and a neutral net charge. ELK-B is a modified ELK peptide that has a 25% bigger hydrophobic phase and +4 net charge.

FIGURE 6.

Comparative structure analysis of peptides exhibiting anti-inflammatory properties and inactive peptides. L37pA has two identical amphipathic helices, called 18A, which are connected with a proline (hydrophobic residues are shown in yellow). The 5A peptide also has two amphipathic helices connected with a proline: 18A and modified 18A, where 5 aa are substituted with alanine, to make this second helix more hydrophobic. Two identical helices of the ELK peptide consist of three types of residues: hydrophobic leucine, positively charged lysine, and negatively charged glutamic acid. Helices have hydrophobic and hydrophilic phases and a neutral net charge. ELK-B is a modified ELK peptide that has a 25% bigger hydrophobic phase and +4 net charge.

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apoA-I, a major structural protein of HDL, ameliorates the development and progression of atherosclerosis by accelerating cholesterol efflux from lipid-laden macrophages (4, 6) and removing proinflammatory oxidized phospholipids from arterial cell walls (4, 38). apoA-I exhibits anti-inflammatory effects in a variety of cell types involved in the pathogenesis of ALI and asthma, such as dendritic cells (39), T cells (40), neutrophils, and macrophages. The protective effects of HDL are due, in part, to high-affinity binding to SR-B receptors, which have recently emerged as important regulators of innate and adaptive immune responses (10, 39, 41). However, pharmaceutical-grade apoA-I protein is expensive and may not be feasible to use as a therapeutic agent (6).

To address this problem, a number of short apoA-I mimetic peptides that retain the beneficial effects of apoA-I have been developed (4, 6). All of these peptides have a helical amphipathic structure enabling their interactions with scavenger receptors including CD36 and its homologs, SR-BI, and SR-BII, as well as others, such as formyl-peptide receptors, LOX-1, and potentially class A scavenger receptors (31, 33, 42). Some of these SAHPs demonstrated anti-inflammatory effects in models of atherosclerosis, cardiac ischemia–reperfusion injury, and endothelial dysfunction (4347), as well as in murine models of adenovirus viral infection (data not shown), endotoxemia (28), sepsis (28), and collagen-induced arthritis (48, 49). Recent data also revealed that one of the SAHPs, 5A, was beneficial in preventing inflammatory and immune responses in a house dust mite–mediated murine model of asthma (50). However, the peptide sequence that exhibits the most potent effects against pulmonary inflammation remains to be determined.

This study used in vitro and in vivo models to assess a panel of apoA-I mimetic peptides with different relative selectivity to CD36 and SR-B group receptors to identify the candidate peptides with most the potent protective properties against LPS-induced lung injury. We generated peptides with different amphipathic and electrostatic properties to test their protective effects against an LPS challenge. Stable cell lines expressing SR-BI, SR-BII, and CD36 receptors were used to analyze the effects of specific receptors and inhibitory peptides on LPS-stimulated IL-8 secretion in vitro and LPS-induced ALI in vivo. The major finding was that selective CD36 antagonists were most effective in protecting against LPS-induced ALI. SR-BI/II selective antagonists were not as effective as CD36 antagonists in LPS-induced ALI in mice, indicating that the SR-BI/II–related anti-inflammatory effects of LKB peptides are not as important as the anti-CD36 effects.

We also demonstrated that SAHPs, such as L37pA, bind to all three SR-B family proteins, including SR-BI, SR-BII, and CD36, in HeLa cells stably transfected with these receptors (Fig. 1). It was demonstrated previously that SAHPs can bind to SR-BI (28), but this is the first report, to our knowledge, showing that L37pA binds to SR-BII and CD36 (Fig. 1). The ability of SR-Bs to respond to inflammatory stimuli was further assessed by measuring IL-8 secretion in epithelial HEK293 cells stably transfected with SR-BI, SR-BII, and CD36. The potency order was CD36 > SR-BII > > SR-BI (31). L37pA was effective in all three cell models and reduced LPS-induced IL-8 secretion by ∼90% (Fig. 2). It also attenuated pulmonary inflammation in LPS-induced ALI (Fig. 3), but L37pA is known to provoke mild erythrocyte hemolysis, which renders it less effective at higher concentrations (25–50 mg/kg) in ALI and cecal ligation and puncture–induced sepsis (Fig. 3) (28). The less toxic 5A was not effective, which prompted us to search the previously developed ELK-based peptide database (Table I). Among >20 peptides, of which most did not discriminate between CD36 and SR-BI/II, only ELK-B and ELK-B2 showed specificity toward CD36 and reduced LPS-induced IL-8 secretion in CD36-expressing HEK cells more significantly than in SR-BI– or SR-BII–expressing HEK cells (Table II). The ELK peptide was the only SAHP that specifically reduced SR-BI/SR-BII–mediated LPS-induced IL-8 secretion in SR-BI and SR-BII HEK293 cells. In vivo analysis showed that L37pA and ELK-B markedly attenuated key parameters of LPS-induced lung dysfunction. ELK-B was also shown in this model to be more potent than 5A. ELK-B and L37pA inhibited the recruitment of PMN cells to the lungs of LPS-challenged mice. Importantly, ELK, an SR-BI/II antagonist, was ineffective in the ALI model, indicating that CD36, rather than SR-BI/II, is of primary importance in LPS-induced ALI.

There are several potential mechanisms underlying the protective effects of SAHPs and ELK-B in the LPS-induced ALI model. First, a key step appears to be the ability of these peptides to bind to SR-Bs and reduce CD36-mediated pulmonary inflammation (Fig. 5). Second, ELK-B can also potentially bind LPS, neutralizing LPS lipid A and preventing the proinflammatory and toxicity effects of LPS (51, 52). However, neutralization effects diminish in the presence of cations, as we demonstrated earlier, and they can be less significant than receptor specificity (33). Third, peptides could affect the LPS-induced effects mediated through other receptors. Previous reports suggested that, in addition to SR-B, SAHPs may bind to other receptors, including formyl peptide receptors, LOX-1, class A scavenger receptors (53), and the TLR family. However, the effect of peptides on TNF- and phorbol ester–induced inflammatory stimulation in various cells, including macrophages, was only marginally affected by SAHPs (54). Furthermore, recent data suggest that CD36 is one of the most important receptors associated with bacterial uptake, bacterial endotoxic cellular cytotoxicity (28), and acute and chronic kidney injury (28, 5557). These recent data suggest that the vicious cycles of pathogen associated molecular pattern–induced inflammation and DAMP-induced tissue damage may well be mediated through CD36 (58, 59). At the same time, independently of the specific mechanisms affecting lung inflammation, SAHPs are important tools that decrease pulmonary damage induced by proinflammatory compounds (51, 60).

In summary, we demonstrated that administration of ELK-B and, to a lesser extent, L37pA, ELK, and 5A attenuated the induction of several key pathogenic features of acute pulmonary damage induced by LPS, including airway inflammation and polymorphonuclear cell migration. These results identify SR-B ligands, SAHPs, and, in particular, ELK-B targeting CD36 as potential novel therapeutics that could be developed to treat pulmonary inflammation and dysfunction in patients who do not respond to standard therapies, such as those with various pulmonary infectious and inflammatory insults.

This work was supported by the National Institutes of Health Intramural Research Programs at the Clinical Center, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute, as well as by National Institutes of Health grants to the Lung Injury Center, The University of Chicago.

Abbreviations used in this article:

ALI

acute lung injury

apoA-I

apolipoprotein A-I

BALF

bronchoalveolar lavage fluid

DAMP

danger-associated molecular pattern

EC

endothelial cell

HDL

high-density LP

HLMVEC

human lung microvascular pulmonary EC

LDL

low-density LP

LOX

lectin-like oxidized low-density LP receptor

LP

lipoprotein

SAHP

synthetic amphipathic helical peptide

SR-B

class B scavenger receptor.

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The authors have no financial conflicts of interest.