Differential Accumulation Patterns for CEP Modifications In Vivo

We first analyzed the pattern of CEP accumulation in wounds on the back of 8-week-old mice for a month. Although CEP was barely detectable in normal skin, its levels were dramatically increased in 3-day-old wounds, following a bell-shaped curve at later time points and returning to the normal levels at day 24 post injury, when wounds were almost completely healed (Figure 1A). Histological analyses revealed that CEP was highly localized in the scabs, granulation tissues, and hypertrophic epidermal wound edges at day 5, whereas at day 24, it was only present in the epidermis and hair follicles (Figure 1B). The transient nature of CEP presence in wounds and its rapid downregulation after completion of angiogenesis and the healing process indicate an existence of a specific mechanism of CEP clearance.

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Figure 1.

Regulation of 2-(ω-carboxyethyl)pyrrole (CEP) levels in vivo. A, The presence of CEP-containing modifications in vivo during wound healing (n=5, top). The wounded skins were collected at the indicated times, homogenized, and then each supernatant was used for enzyme-linked immunosorbent assay (ELISA). CEP concentration was normalized by total protein concentration. Bottom, Representative images of wounds on the mouse back skins at the times indicated (n=5) are shown. B, Hematoxylin and eosin (H/E) and CEP immunofluorescence staining of the wounded skins at day 5 (5d) and 24 (24d). Note that CEP is stained on the scabs, granulation tissues, and hypertrophic epidermal wound edges at day 5, whereas CEP is slightly stained in the epidermis and hair follicles at day 24. C, CEP accumulation in implanted B16-F10 murine melanomas was measured by ELISA. Melanomas were collected at day 7, 14, and 21 after subcutaneous injections of B16-F10 murine melanoma cells (7.0×10⁵ cells per injection) into mice (n=4). D, Hematoxylin and eosin and fluorescence staining for CEP, CD68 (macrophages), and 4′,6-diamidino-2-phenylindole (DAPI, nuclei) of the aorta of Apoe^−/− mice (fed with Western diet [WD] for 5 weeks) and age- and gender-matched wild-type (WT) control mice (fed with chow diet [CD]). Note that CEP was positively stained only in atherosclerotic lesions (n=3). Bottom, Quantification of CEP and CD68 immunofluorescence of WT and Apoe^−/− mice. Fold increase over WT control is shown. E, Plasma levels of CEP from the same Apoe^−/− and WT mice (D), measured by ELISA (n=3). Fold increase over WT control is shown. The absolute concentration of CEP in Apoe^−/− mice was 70–100×10^–12 mol/L based on CEP standard (see Methods). F, Plasma levels of CEP from Apoe^−/− and WT mice, which were fed with a CD or WD for 16 weeks, were measured by ELISA. Fold increase over WT control on CD is shown. G, Upregulation of tumor necrosis factor-α (TNF-α), measured by ELISA, in thioglycolate-elicited mouse peritoneal macrophages on treatment with 10 μmol/L of CEP-bovine serum albumin (BSA) for 4 hours. H, Upregulation of Tnf-α gene, measured by quantitative polymerase chain reaction, in resident mouse peritoneal macrophages on treatment with 10 μmol/L of CEP-BSA for 4 hours. Fold increase over BSA-treated cells is shown. *P<0.05, **P<0.01, ***P<0.005.

Because wound healing is a physiological process that restores original tissue integrity and functionality, we next assessed CEP accumulation in chronic pathological processes associated with continuous oxidative stress, such as tumor progression and atherosclerotic lesion development.^4,30 CEP levels in melanomas were continuously increasing during tumor progression at 7, 14, and 21 days post implantation, whereas the tumor volume was increased by >60-fold within 2 weeks (Figure 1C; Online Figure I). Likewise, CEP was present at high levels in atherosclerotic lesions of Apoe^−/− mice fed with a Western diet (WD) when compared with a control, in which there was no positive signal for CEP (Figure 1D). Moreover, the plasma CEP levels in these Apoe^−/− mice were increased >8-fold when compared with controls, demonstrating that CEP presence in vivo was not limited to the atherosclerotic lesions but was a rather systemic response (Figure 1E). Plasma CEP levels in Apoe^−/− mice were higher than those of wild-type (WT) mice even without a WD, however, a WD substantially augmented the oxidative processes resulting in at least 2-fold increase of CEP levels in blood of Apoe^−/− mice (Figure 1F). After 18 weeks on a WD, CEP levels within the atherosclerotic lesions of Apoe^−/− mice remained continuously high (Online Figure II). Thus, the transient nature of CEP presence during wound healing is in contrast to its continuous accumulation in pathologies associated with inflammation and oxidative stress, implying the existence of a specific mechanism responsible for CEP degradation.

Besides the proangiogenic effect of CEP (Online Figure III),^14,23 it has a proinflammatory role.^21,22,24 Indeed, in the presence of CEP, macrophages upregulated a proinflammatory cytokine, tumor necrosis factor-α, both at a protein (Figure 1G) as well as at an mRNA level (Figure 1H). Together with previous observations, this suggests that high levels of CEP might augment chronic pathological processes and emphasize an importance of the timely removal of this end product of LPO.

CEP Binding to Macrophages

To identify the cell type responsible for CEP clearance, we first tested the ability of various immune cells, such as R-MPMs (resident mouse peritoneal macrophages), bone marrow neutrophils, and lymph node T and B cells to bind to CEP directly. Based on fluorescence-activated cell sorting analysis, macrophages bound to CEP-bovine serum albumin (BSA) in a concentration-dependent manner, achieving saturation at 200 nmol/L (Figure 2A). Then, we observed 2 distinct populations of macrophages according F4/80 expression, F4/80^lo and F4/80^hi, and measured CEP binding by fluorescence-activated cell sorting. F4/80^hi macrophages bound to CEP in a concentration-dependent manner and the maximum binding of CEP-BSA to this cell population was at least 10× higher than to F4/80^lo macrophages based on fluorescence intensity (Figure 2B). F4/80^lo macrophages are a minor subset of R-MPMs, whereas >80% of total R-MPMs are F4/80^hi (Online Figure IVA). MHC-II (major histocompatibility complex class II) expression is high on F4/80^lo macrophages, which are known to serve as antigen-presenting cells in vitro, but low on F4/80^hi macrophages (Online Figure IVB). In contrast, scavenger receptors and pattern recognition receptors, such as CD36 and TLR2, respectively, are highly expressed on F4/80^hi macrophages (Online Figure IVB), which are known to be involved in phagocytosis of apoptotic cells in vivo.²⁸ The concentration-dependent manner of CEP-BSA binding with a clear saturation together with differential interaction with distinct cell populations suggests the involvement of specific cell surface receptors. To rule out the possibility that the binding might be mediated by BSA but not by the CEP moiety, fluorescein isothiocyanate (FITC)-conjugated BSA (FITC-BSA) was used as a control. Fluorescence-activated cell sorting analysis showed that there were dose-dependently positive signals when CEP-BSA, but not FITC-BSA, was exposed to macrophages (Online Figure V).

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Figure 2.

2-(ω-carboxyethyl)pyrrole (CEP) binding and scavenging by macrophages. A and B, Flow cytometry analysis of CEP binding on primary blood cells, such as macrophages, neutrophils, T and B cells (A), and F4/80^hi and F4/80^lo macrophages (B), which were incubated on ice for 30 minutes with CEP-bovine serum albumin (BSA) at the concentrations indicated, after freshly isolated from mouse bone marrow (for neutrophils), lymph nodes (for T and B cells), and peritoneum (for macrophages and B cells). Mean fluorescence intensity was normalized to that of 300 nmol/L CEP treatment, which was assigned a value of 100%. C, Immunofluorescence staining for CEP, CD68, and 4′,6-diamidino-2-phenylindole (DAPI) on the frozen sections of mouse back skins collected at 2 hours after intradermal injections of 10 μmol/L CEP-BSA or an equal amount of BSA as a control. Right, CEP presence in cells was measured by quantification of CEP/DAPI staining, whereas CEP presence specifically in macrophages was assessed based on CEP/CD68 costaining in tissues section (left). The value of CEP/DAPI costaining was assigned 100%. D and E, Immunostaining for CEP, CD68, and DAPI on the primary resident mouse peritoneal macrophages (R-MPMs) is shown. Cells were incubated at 37°C for the times indicated; then washed, culture media were changed with fresh media without CEP-BSA, and incubated for additional 0, 30, and 90 minutes after 30 minutes for pulse treatment (D) or during the entire incubation (E) with 500 nmol/L of CEP-BSA. F, CEP was quantified by enzyme-linked immunosorbent assay (ELISA) before and after incubation with macrophages. After 500 nmol/L CEP-BSA was added to the R-MPMs at 37°C, the supernatants were then collected at the times indicated, measured by ELISA, and normalized to the CEP only-containing culture media without macrophages. Percentage of remaining CEP is shown. Initial concentration of CEP (time point 0) was assigned a value of 100%. G, Simplified chemical structures of CEP and EP. H, Detection of ethylpyrrole (EP) by immunofluorescence in primary R-MPMs. In parallel, the staining for CD68 and DAPI is shown. R-MPMs were incubated at 37°C for the times indicated with 500 nmol/L of EP-BSA. Note that certain levels of EP are endogenously present in cells, but both binding and scavenging are lacking. Right, Quantification of EP fluorescence at the times indicated. Fold changes in EP staining are shown. Initial staining for EP (0 hours) was assigned a value of 1. I, Immunofluorescence of F4/80 (macrophage) and DAPI on the back skins of mice treated with control- or clodronate-liposome for 3 days. Right, Quantification of F4/80 fluorescence based on tissue sections shown (left). Fold changes in macrophage count is shown. Macrophage count in mice treated with control antibody was assigned a value of 100%. J, Impaired CEP clearance in clodronate-treated mice (as in I). Skins were collected at the times indicated from control- or clodronate-liposome treated mice (n=3), after subcutaneous injections of 10 μmol/L CEP-BSA. Concentration of CEP in homogenates was measured by ELISA. Fold changes in CEP levels are shown. CEP concentration in skin 1 hour post injection was assigned a value of 1. ***P<0.005.

Next, we assessed whether exogenously provided CEP-BSA can interact with macrophages in vivo. Two hours after CEP injection, >80% of cells positive for CEP were also positive for CD68, a macrophage marker (Figure 2C). Thus, CEP is recognized by macrophages in vitro as well as in vivo.

Macrophages Are Able to Specifically Scavenge CEP But Not Similar Protein Modifications

To examine whether macrophages are able to scavenge and metabolize CEP, R-MPMs (that do not generate their own CEP) were incubated with exogenous CEP, washed, and the presence of remaining metabolite in cells was monitored. Within the first 30 minutes of incubation, CEP was strongly present in R-MPMs, however, the CEP positive signal was gradually reduced at 30 minutes and finally disappeared at 90 minutes after media change, indicating that CEP was being scavenged by macrophages (Figure 2D). When macrophages were incubated with CEP for 24 hours without media change, all of the CEP had merged with CD68, known to be expressed in the lysosomes^31,32 (Figure 2E), implying that CEP was scavenged and possibly metabolized by macrophages. The rate of CEP scavenging by macrophages was monitored by measuring the concentration of CEP remaining in the culture medium by ELISA. As macrophages actively bound and scavenged CEP, the CEP levels reached at approximately 70% of the initial concentration within 30 minutes and continued decreasing to approximately 20% of the original CEP amount within 24 hours (Figure 2F).

To investigate the specificity of CEP recognition and clearance, we compared it with that of a structurally similar protein modification, ethylpyrrole (EP). This protein modification is not only structurally similar to CEP (only lacking the carboxy group as shown in Figure 2G), but it is cogenerated through an alternative oxidative cleavage of DHA to give 4-hydroxyhex-2-enal followed by condensation of 4-hydroxyhex-2-enal with the ε-amino group of lysyl residues (Online Data Supplement; Online Figure VIA). We compared the interaction of EP-BSA and CEP-BSA with macrophages (Online Figure VIB). As shown in Figure 2H, EP-BSA exhibited no binding to macrophages, which were not able to scavenge it for as long as 24 hours. Therefore, it seems that the process of recognition and scavenging by macrophages is highly specific for CEP-containing derivatives and it is not applicable to similar derivatives, such as EP-BSA modifications.

To demonstrate whether and how CEP is scavenged by macrophages in vivo, macrophages were depleted by clodronate treatment,^33,34 resulting in at least 50% decrease in the amount of resident macrophages in skin (Figure 2I). Exogenous CEP was subcutaneously injected into control and macrophage-depleted mice, and amount of CEP in vivo was monitored. Although CEP levels in control mice were decreased in a time-dependent manner reaching ≈60% and 40% after 3 and 6 hours, respectively, there was a substantial delay in macrophage-depleted mice, where the levels were unchanged for at least 3 hours after CEP injection (Figure 2J). At the 6-hour time point, the remaining amount of CEP was about 2× higher in macrophage-depleted mice when compared with controls (Figure 2J). Collectively, these results demonstrate that CEP, a stable end-product of DHA oxidation, is scavenged by macrophages ex vivo and in vivo.

M2-Like Macrophages Efficiently Bind to and Scavenge CEP

As macrophages are mainly classified into 2 subtypes, M1 and M2, we examined whether 1 of these 2 populations preferentially bind to and scavenge CEP. To polarize macrophages toward M1- or M2-phenotypes, R-MPMs were treated by interferon-γ and lipopolysaccharide or interleukin-4, respectively, and the polarization was verified using known M1- or M2-markers.²⁷ Although M1-markers, Nos2 and Cxcl10, were remarkably increased in the M1-like macrophages; M2-markers, Chi3l3 and Cd36, were markedly increased on the M2-like macrophages (Figure 3A). Although M2-like macrophages were able to efficiently bind CEP, the M1-like macrophages exhibited relatively low binding (Figure 3B). CEP scavenging activity was also significantly lower for the M1-like macrophages compared with the M2-like macrophages (Figure 3C). These results indicate that both, CEP binding and scavenging, were primarily mediated by M2-like macrophages rather than by M1-like macrophages.

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Figure 3.

2-(ω-carboxyethyl)pyrrole (CEP) binding and scavenging by M1- or M2-like macrophages. A, Expression of M1 and M2 marker genes in polarized macrophages. Quantitative real-time polymerase chain reaction analysis of representative M1 marker genes (Nos2 and Cxcl10) and M2 marker gene (Chi3l3 and Cd36) normalized by 18s, from resident mouse peritoneal macrophages (R-MPMs) is shown. B, Immunofluorescence of CEP, inducible nitric oxide synthase (iNOS, M1 marker), and 4′,6-diamidino-2-phenylindole (DAPI) on M1-like and M2-like macrophages treated by 500 nmol/L of CEP-bovine serum albumin (BSA) for 20 minutes. C, CEP levels that remained in the culture media. After 500 nmol/L CEP-BSA was treated to the R-MPMs at 37°C for 24 hours, the supernatants were collected. CEP levels were measured by enzyme-linked immunosorbent assay and normalized to the CEP only-containing culture media without macrophages (this was assigned a value of 100%). **P<0.01, ***P<0.005.

CEP Demonstrates Direct Interactions With CD36 and TLR2

As CEP was shown to bind to TLR2 on endothelial cells to induce angiogenesis, we examined whether TLR2 on macrophages serves as a possible receptor for CEP. Because TLR2 is known to partner with other receptors, such as CD14 and CD36, these receptors were also considered. CD36 is highly expressed by both F4/80^hi- and M2-like macrophages and is known to recognize several ligands, including modified lipoproteins, glycated proteins, and amyloid-forming peptides (Figure 3A; Online Figure IVD^35–37). Accordingly, we assessed CEP interactions with recombinant TLR2-Fc, CD36-Fc, and CD14-Fc chimeras in a solid phase immunoassay. Both, CD36 and TLR2, bound to CEP in a concentration-dependent manner, whereas binding to CD14-Fc and Fc fragment alone was negligible (Figure 4A). Then, the binding of CEP to CD36 and TLR2 was measured in real time by surface plasmon resonance where CEP was injected and allowed to flow over immobilized CD36-Fc or TLR2-Fc (Figure 4B). The binding was confirmed by reversed surface plasmon resonance, where CD36-Fc or TLR2-Fc protein was injected at various concentrations and allowed to flow over immobilized CEP. Consistent with the results from Figure 4A, both proteins bound to CEP (Figure 4C and 4D). The dissociation constants (Kd) for CD36 and TLR2 with CEP were 6.53 and 11.8 nmol/L, respectively. Since the functions of TLR2 depend on its association with either TLR6 or TLR1, where TLR6 is known to be involved in its partnership with CD36,³⁸ we assessed the direct binding of CEP to both TLRs using surface plasmon resonance. Although CEP interacted with both receptors (Online Figure VIIA–VIIC), the binding to TLR6 was substantially higher than that to TLR1 and compatible to that to CD36. This was confirmed by ELISA (Online Figure VIID). CEP binding to Tlr6^−/− macrophages and the consequent intake was diminished compared with WT cells as evidenced by ≈30% decrease in average fluorescence intensity (Online Figure VIIE and VIIF).

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Figure 4.

Direct binding between 2-(ω-carboxyethyl)pyrrole (CEP) and CD36 or toll-like receptor 2 (TLR2). A, Enzyme-linked immunosorbent assay–based binding assay using various concentrations of CEP-bovine serum albumin (BSA) (0–1600 nmol/L) and Fc-conjugated chimeric proteins, such as TLR2-Fc, CD36-Fc, CD14-Fc. Binding to Fc-only served as a negative control (control-Fc). The absorbance values at 415 nm are shown. Values obtained using BSA-alone (as a coating substrate) were subtracted. B–D, Surface plasmon resonance (SPR) analysis of the binding between CEP and CD36 (B, C, E, and F), CEP and TLR2 (B and D). CD36-Fc, TLR2-Fc, and Fc-only were immobilized on CM5 chip and 2.5 μmol/L CEP-human serum albumin (HSA) in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer was flowed over the chip (B). CEP-HSA was immobilized on a CM5 chip and various concentrations of CD36-Fc (1.4–140 nmol/L; C) or TLR2-Fc (1–200 nmol/L; D) were flowed over the chip. Note that CEP demonstrates direct binding to CD36 and, to a lesser extent, TLR2. E and F, SPR analyses for CEP binding site on CD36. CD36-Fc (70 nmol/L) were preincubated with various concentrations of copper oxidized low-density lipoprotein (OxLDL; 10–200 μg/mL) and flowed over immobilized CEP-HSA. CEP binding to CD36 in the absence of OxLDL was assigned a value of 100% (E). Various concentrations of CD36 peptide (¹⁶⁰SLINKSKSSMF¹⁷⁰) or scrambled peptide (SIKVNLSQMKILNSI) were preincubated with 10 μmol/L of CEP-HSA and flowed over immobilized CD36-Fc. CEP binding to CD36 in the absence of peptide was assigned a value of 100% (F).

To further characterize the binding site for CEP on CD36, we performed a competition assay using a known ligand for CD36, oxidized low-density lipoprotein.³⁶ Oxidized low-density lipoprotein competed with CEP in a dose-dependent manner, indicating that CEP and oxidized low-density lipoprotein might share the same binding site on CD36 (Figure 4E). Indeed, ¹⁶⁰SLINKSKSSMF¹⁷⁰ peptide derived from an extracellular domain of human CD36 and known to be critical for oxidized low-density lipoprotein binding³⁹ inhibited CEP–CD36 interaction in a dose-dependent manner (Figure 4F).

CEP Scavenging Depends on CD36 and TLR2

The physical binding of CEP to CD36 was further confirmed by 2 different cell-based binding assays using stable cell lines expressing CD36, Raw264.7-CD36, and HEK293-CD36 (Online Figure VIII). HEK293-CD36 cells bound about 3× more to CEP than control cells (Figure 5A). A similar trend was observed using Raw264.7-CD36 cells (Figure 5B).⁴⁰

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Figure 5.

2-(ω-carboxyethyl)pyrrole (CEP) binding and scavenging by CD36 and toll-like receptor 2 (TLR2). A and B, Cell-based CEP binding assay using HEK293 (A) and Raw264.7 (B), which were stably expressing CD36. CEP binding to the cells was visualized by fluorescence-activated cell sorting (FACS) after staining the cells with rabbit anti-CEP antibody and Alexa-488-conjugated antirabbit IgG secondary antibody. Mean fluorescence intensity are shown. C, Reduced CEP binding to CD36 null cells. Immunofluorescence of CEP, CD68, and 4′,6-diamidino-2-phenylindole (DAPI) on the primary wild-type (WT) and Cd36^−/− resident mouse peritoneal macrophages (R-MPMs), which were incubated at 37°C with 500 nmol/L CEP-BSA for 30 minutes. D, Reduced CEP scavenging by CD36 null cells. WT and Cd36^−/− R-MPMs were treated with 500 nmol/L CEP-bovine serum albumin (BSA) for 24 hours at 37°C, then the supernatants were collected. CEP levels were measured by enzyme-linked immunosorbent assay (ELISA) and residual CEP values are shown. The amount of CEP without added R-MPMs was assigned a value of 100%. E, Reduced CEP binding to TLR2 null cells. Immunofluorescence of CEP, CD68, and DAPI on the primary WT and Tlr2^−/− R-MPMs, which were incubated at 37°C with 500 nmol/L CEP-BSA for 30 minutes. F, CEP scavenging by Tlr2^−/− cells is reduced. WT and Cd36^−/− R-MPMs were treated with 500 nmol/L CEP-BSA for 24 hours at 37°C, then the supernatants were collected. CEP levels measured by ELISA are shown. Amount of CEP without added R-MPMs was assigned a value of 100%. ***P<0.005.

Then, the role of CD36 in CEP clearance was tested using CD36-null (Cd36^−/−) macrophages. When treated with CEP for 30 minutes, Cd36^−/− macrophages bound substantially less CEP than WT macrophages (Figure 5C). Moreover, Cd36^−/−- macrophages were defective in their ability to scavenge CEP. Although WT macrophages were able to metabolize about 40% of CEP in first 6 hours, CEP levels were not significantly changed in the presence of Cd36^−/− macrophages (Figure 5D). After 24 hours, WT macrophages were able to remove ≈85%, whereas Cd36^−/− macrophages metabolized only 40% of exogenously provided CEP (Figure 5D), indicating that CD36 on macrophages is essential for CEP binding and scavenging.

Similar experiments were conducted using Tlr2^−/− macrophages. Compared with WT, Tlr2^−/− macrophages bound less to CEP within 30 minutes (Figure 5E). Accordingly, CEP scavenging activity was also reduced in Tlr2^−/− macrophages since after 24 hours, remaining amounts of CEP were ≈15% for WT and 40% for Tlr2^−/− macrophages (Figure 5F). These experiments demonstrate that similar to CD36, TLR2 deficiency affects CEP binding and scavenging by macrophages, although the role of CD36 seems to be more prominent.

CEP Clearance Is Severely Impaired in CD36/TLR2 DKO Macrophages

Because CD36 often serves as a coreceptor for TLR2,^38,41 we generated Cd36^−/−;Tlr2^−/− double knockout (DKO) mice to assess the cooperation between these receptors in CEP clearance by macrophages. We compared the ability of R-MPMs from WT, Cd36^−/−, Tlr2^−/−, and DKO mice to bind to and scavenge CEP. Although both, Tlr2^−/− and Cd36^−/−, macrophages bound only 60% and 75% less to CEP than WT, respectively, the binding to DKO macrophages was reduced by about 90% when compared with WT (Figure 6A and 6B). Likewise, the amount of CEP scavenged by WT, Tlr2^−/−, Cd36^−/−, and DKO macrophages within 24 hours was 70%, 55%, 40%, and 22%, respectively (Figure 6C). Together, the additive effect of Tlr2^−/− and Cd36^−/− deficiencies suggests that while these receptors are both important for CEP binding and clearance by macrophages, they might function independently and the role of CD36 was more prominent.

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Figure 6.

Additive effect of CD36 and toll-like receptor 2 (TLR2) on 2-(ω-carboxyethyl)pyrrole (CEP) binding and scavenging. A, Immunofluorescence of CEP, CD68, and 4′,6-diamidino-2-phenylindole (DAPI) using primary WT, Tlr2^−/−, Cd36^−/−, and Cd36^−/−;Tlr2^−/− double knockout (DKO) resident mouse peritoneal macrophages (R-MPMs), which were incubated at 37°C with 500 nmol/L CEP-bovine serum albumin (BSA) for 30 minutes. B, Quantification of CEP binding based on fluorescence. Binding to wild-type (WT) macrophages was assigned a value of 100% (A). C, Tlr2^−/−, Cd36^−/−, and Cd36^−/−;Tlr2^−/− DKO R-MPMs were treated with 500 nmol/L CEP-BSA for 24 hours at 37°C, then the supernatants were collected. CEP levels were measured by enzyme-linked immunosorbent assay and residual CEP values are shown. The amount of CEP without added R-MPMs was assigned a value of 100%. *P<0.05, **P<0.01, ***P<0.005.

Defective CEP Clearance Affects Angiogenesis and Wound Closure in CD36 Null Mice

To assess the importance of CEP presence and timely removal in tissues, we analyzed angiogenesis and wound healing in Cd36^−/− mice. Punch wounds were made on the back skin of 8-week-old WT and Cd36^−/− mice and monitored for 7 days. Surprisingly, at the early time points (day 3), wound closure was accelerated in Cd36^−/− mice when compared with WT, albeit no longer significantly at day 7 (Figure 7A; Online Figure IXA), which might be because of delayed clearance of apoptotic cells also mediated by CD36.³⁵ At the same time, CD31 immunostaining revealed that angiogenesis in Cd36^−/− wounds also occurred earlier than in WT mice (day 3 versus days 5–7, respectively; Figure 7B; Online Figure IXB). Costaining for CD31 and CEP revealed the presence of new blood vessels primarily in the CEP-positive area, suggesting that the faster angiogenesis might be because of increased levels of CEP in the Cd36^−/− wounds (Figure 7B).

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Figure 7.

Defective 2-(ω-carboxyethyl)pyrrole (CEP) clearance in Cd36^−/− wounds. A, Representative images of wounds on the back skins of wild-type (WT) and Cd36^−/− mice at day 3 (n=10). B, Two independent representative immunofluorescence staining for CEP, CD31, and 4′,6-diamidino-2-phenylindole (DAPI) in tissue sections of WT and Cd36^−/− mice at day 3 after wounding (n=5). Bottom, Quantification of CEP and CD31 fluorescence in tissue section of WT and Cd36^−/− mice normalized to WT control is shown. C, Representative images of wounds on the back skins of Cd36^−/− mice treated with either control- or CEP blocking antibody at day 3 (n=4). D, Immunofluorescence of CD31 and DAPI in skin of Cd36^−/− mice treated with either control- or CEP blocking antibody at day 3 after wounding (n=4). Bottom, Quantification of CD31 fluorescence normalized to WT control. E, Immunofluorescence of CEP, F4/80, and DAPI in tissues sections of WT and Cd36^−/− mice at day 3 after wounding. Right, Enlarged pictures of single macrophages (arrowheads). F and G, Multiphoton confocal images of CEP, F4/80, and DAPI staining of the single cells from wounded skins of WT and Cd36^−/− mice at day 3 show reduced CEP levels in Cd36^−/− skins. G, A cross-sectional image of WT cell and a side view of Cd36^−/− cell were obtained from 3-dimensional reconstructions of multiphoton confocal z-stack images. Note that CEP localization differs between WT and Cd36^−/− macrophages: it is seen in WT cells in cross-sectional images (inside of the cells) but not in other planes, whereas its presence was mainly extracellular in Cd36^−/− cells. *P<0.05, **P<0.01, ***P<0.005.

To demonstrate the causative effect of CEP accumulation on the accelerated angiogenesis and wound closure in Cd36^−/− mice, a mouse anti-CEP monoclonal antibody was introduced during wound healing to block the proangiogenic activity of CEP in vivo. The blockade of CEP activity in Cd36^−/− mice substantially inhibited both the accelerated angiogenesis and wound closure, indicating a causative role for CEP accumulation in those processes (Figure 7C and 7D). Despite the comparable numbers of recruited macrophages, CEP clearance was less efficient in Cd36^−/− wounds when compared with WT, based on F4/80 and CEP staining (Figure 7E). Analysis of the single cells in WT and Cd36^−/− wounds at day 3 by a multiphoton confocal microscopy revealed that CEP was localized inside of the cells in WT, but remained on the cell membrane in Cd36^−/− macrophages, implying defective scavenging (Figure 7F and 7G). Taken together, these results demonstrate that CD36 is absolutely required for CEP clearance in vivo. Cd36^−/− mice exhibit increased CEP accumulation, which, in turn, mediates the accelerated angiogenesis and wound closure.