Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor β–Expressing Macrophages

Background Folate receptor β (FRβ) is induced during macrophage activation. A recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to an anti-FRβ antibody (anti–FRβ-PE38) has been reported to kill activated macrophages in inflammatory diseases. To elucidate the effect of an immunotoxin targeting FRβ on atherosclerosis, we determined the presence of FRβ-expressing macrophages in atherosclerotic lesions and administered the FRβ immunotoxin in apolipoprotein E–deficient mice. Methods and Results The FRβ-expressing macrophages were observed in atherosclerotic lesions of apolipoprotein E–deficient mice. At 15 or 35 weeks of age, the apolipoprotein E–deficient mice were divided into 3 groups and were intravenously administered 0.1 mg/kg of anti–FRβ-PE38 (immunotoxin group), 0.1 mg/kg of PE38 (toxin group), or 0.1 mL of saline (control group) every 3 days, for a total of 5 times for each age group. The mice were analyzed at 21 or 41 weeks of age. Treatment with the immunotoxin resulted in 31% and 22% reductions in atherosclerotic lesions of the 21- and 41-week-old mice, respectively (P<0.05). Administration of immunotoxin reduced the numbers of FRβ- and tumor necrosis factor-α–expressing macrophages, reduced cell proliferation, and increased the number of apoptotic cells (P<0.05). Real-time polymerase chain reaction demonstrated that the expression of FRβ and tumor necrosis factor-α mRNA was significantly decreased in the immunotoxin group (P<0.05). Conclusions These results suggest that FRβ-expressing macrophages exist in the atherosclerotic lesions of apolipoprotein E–deficient mice and that FRβ immunotoxin administration reduces the progression of atherosclerotic lesions in younger and older individuals. The recombinant FRβ immunotoxin targeting activated macrophages could provide a novel therapeutic tool for atherosclerosis. (J Am Heart Assoc. 2012;1:e003079 doi: 10.1161/JAHA.112.003079.)

A therosclerosis is a chronic inflammatory disease in which activated macrophages play an important role by producing cytokines, chemokines, proteolytic enzymes, and growth factors. 1,2 Induction of macrophage apoptosis during the early stage of atherosclerosis reduces lesion progression. 3 In contrast, induction of macrophage apoptosis in the advanced stage contributes to the lipid core of an atheroma and leads to the progression of atherosclerotic lesions. 4,5 Thus, the antiatherogenic effect of inducing macrophage apoptosis is controversial because the consequences of macrophage apoptosis differ according to the stage of the lesion. 6 A folate receptor (FR) is a glycoprotein that is anchored to various cells by a glycosylphosphatidylinositol linkage. 7 The FR exhibits a high affinity for folic acid and mediates its transmembrane transport into the cytoplasm for biosynthesis of purine and pyrimidine. 8 The FR has at least 3 isoforms (a, b, c). 9,10 Previously, we showed that FRb is induced during macrophage activation and that FRb expression is a characteristic of activated macrophages. 11 Furthermore, we demonstrated that a recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to a disulfide-stabilized fragment of the variable region of an anti-FRb antibody (dsFv anti-FRb-PE38) killed activated macrophages in rheumatoid arthritis and experimental models of pulmonary fibrosis. 12,13 It recently has been reported that because the FR is a marker of macrophage activation, FR-expressing macrophages represent a specific target for folate-linked drugs, given that they would not be taken up by nonactivated macrophages. 14 It also has been reported that macrophages taking up folate accumulate in the atherosclerotic lesions of apolipoprotein E (apoE)-deficient mice. 15 We therefore hypothesized that depletion of FRb-expressing macrophages via the dsFv anti-FRb-PE38 immunotoxin could be beneficial in treating atherosclerosis by specifically targeting activated macrophages.
The purpose of the present study was to determine the presence of FRb-expressing macrophages in atherosclerotic lesions and to investigate the effect of the recombinant anti-FRb-PE38 immunotoxin administered at an early or late stage of atherosclerosis in a murine model of experimental atherosclerosis.

Methods Experimental Animals
ApoE-deficient mice on a C57BL/6J background were kindly provided by Dr Jan L. Breslow (Rockefeller University, New York, NY). The mice were maintained in a temperature-controlled facility and fed a regular mouse chow diet. All animal procedures were performed in accordance with the Ethical Guideline for Animal Experiments, Kagoshima University, and were approved by the Kagoshima University Committee. The study conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Production of Anti-Mouse FRb Monoclonal Antibodies
Rat anti-mouse monoclonal antibodies (mAbs) against FRb were produced in our laboratory as previously described. 12,16 Briefly, mouse FRb complementary DNA (cDNA) was prepared from a reverse transcription-polymerase chain reaction (PCR) product derived from BALB/c mouse liver. Primer sequences used were as follows: 5′-TCTAGAAAGACATGGCCTGGAAA-CAG-3′ (upstream) and 5′-CCCAACATGGATCAGGAACT-3′ (downstream). Wistar-Kyoto rats (Charles River Laboratories, Yokohama, Japan) were immunized with RBL2H3 (rat mastocytoma) cells transfected with the mouse FRb gene. Lymphocytes from the iliac lymph nodes were fused with NS-1 myeloma cells. Hybridomas were screened for reactivity with FRb-transfected RBL2H3 cells by flow-cytometric and Western blot analyses.

Production of a Recombinant Immunotoxin Against Mouse FRb
The recombinant dsFv anti-FRb-PE38 immunotoxin, which consists of the immunoglobulin (Ig) heavy-chain Fv portion of the anti-mouse FRb mAb (IgV L ) with PE38 (V H -PE38) and the Ig light-chain Fv portion of anti-mouse FRb mAbs, was prepared as previously described. 16,17 In brief, inclusion bodies from bacteria transfected with expression plasmids encoding the IgV H -PE38 and IgV L genes were solubilized separately and then combined. Properly folded dsFv anti-FRb-PE38 was purified by anion-exchange chromatography and size-exclusion chromatography. The half-maximal inhibitory concentration 50 of the immunotoxin, which was determined by the decrease in propidium iodine staining, was 10 ng/mL and 100 ng/mL for FRb-transfected B300-19 cells (a murine pre-B cell line) and thioglycollate-elicited peritoneal macrophages from BALB/c or C57BL/6J mice, respectively. The recombinant immunotoxin contained <5 endotoxin units per milligram.

Experimental Protocol of Immunotoxin Administration in a Mouse Model of Atherosclerosis
We analyzed the effects of the recombinant immunotoxin on atherosclerosis in the male apoE-deficient mice ( Figure 1). We divided the 15-and 35-week-old apoE-deficient mice into 3 groups: immunotoxin, toxin, and control (15 weeks: n=8 per group; 35 weeks: n=5 per group). During a period of 2 weeks, the immunotoxin group intravenously received 0.1 mg/kg of dsFv anti-FRb-PE38 in 100 lL of saline, the toxin group received 0.1 mg/kg of PE38 in 100 lL of saline, and the control group received 100 lL of saline every 3 days, for a total of 5 times for each age group. The atherosclerotic lesions in the aortic roots were analyzed 1 week and 4 weeks later.

Tissue Preparation and Lesion Assessment
The apoE-deficient mice were anesthetized with pentobarbital (80 lg/kg body weight, intraperitoneally), and blood samples were collected from the left ventricle. The number of leukocytes was counted by using a Coulter counter. Plasma cholesterol levels were measured with the use of an enzymatic kit (Kainos Laboratories, Tokyo, Japan). After intraventricular perfusion with 0.9% saline, the heart and ascending aorta were removed and quickly frozen in liquid nitrogen. Hearts were embedded in the Tissue-Tek OCT compound (Sakura Finetek Japan Co, Ltd, Tokyo, Japan), and 5-lm-thick cross sections of aortic roots were prepared, from the aortic valve leaflets through to the end of the atherosclerotic lesion. Every tenth section (50 lm apart) was stained with Oil Red O and hematoxylin. The atherosclerotic lesions were determined as the percentage of the lipid-stained area per vessel area, as described previously. 18,19 Immunohistochemistry Immunohistochemical staining was performed as described previously. 20,21 Acetone-fixed frozen sections (5 lm thick) were stained with a monoclonal rat anti-mouse CD68 antibody (AbD Serotec, Oxford, UK), monoclonal rat anti-mouse FRb antibody, polyclonal rabbit anti-mouse tumor necrosis factor-a (TNF-a) antibody, a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), rabbit anti-mouse CD31 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human CD3 (Dako, Kyoto, Japan), or isotype-matched irrelevant antibodies (AbD Serotec). After blocking with 3% skimmed milk, endogenous peroxidase activity was quenched with 0.3% H 2 O 2 in methanol for 10 minutes, and slides were incubated overnight with a primary antibody at 4°C in a moisture chamber. Staining was performed with a MAX-PO secondary antibody (Nichirei Co, Ltd, Tokyo, Japan) and the NovaRed kit (Vector Laboratories, Burlingame, CA). Morphometric analysis was performed with a Zeiss Axioskop microscope (Carl Zeiss, Jena, Germany) and with Image J analysis software, and the percentage of plaque area exhibiting antibody staining was measured at 21 and 41 weeks of age. Double immunofluorescence staining was performed on the frozen tissue sections. First, sections were reacted with the rat anti-FRb mAb followed by the chicken anti-rat IgG-conjugated Alexa Fluor 594 (Molecular Probes, Eugene, OR). Sections then were incubated with one of the following: (1) Alexa Fluor 488conjugated rat anti-mouse CD68 mAb (AbD Serotec, Raleigh, NC); (2) rabbit anti-mouse CD31 (Santa Cruz Biotechnology); (3) rabbit anti-human CD3 (Dako), followed by the goat antirabbit IgG-conjugated Alexa Fluor 488 (Molecular Probes); or (4) mouse anti-mouse SMA (Santa Cruz Biotechnology), followed by the goat anti-mouse IgG-conjugated Alexa Fluor 488 (Molecular Probes). Immunofluorescent double-stained sections were imaged with an LSM-700 confocal laser microscope (Carl Zeiss, Jena, Germany).

Measurement of Apoptotic Cells
One week after the last injection, terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) was performed to detect apoptotic cells. Five-micrometer-thick cryostat sections were fixed in acetone, underwent staining with the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer's instructions, and were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Control slides were treated with incubation buffer that did not contain the terminal deoxynucleotidyl transferase (TdT) enzyme. The TUNEL-positive cells were counted by using a BZ-8000 fluorescence microscope (Keyence, Osaka, Japan), and the percentage of cells undergoing apoptosis was calculated from the mean number of TUNELpositive cells and the mean total number of cells from 3 randomly chosen fields from each section (18 weeks: n=8; 38 weeks: n=5).

In Vitro Effect of FRb Immunotoxin
Male apoE-deficient mice (n=3) at 12 weeks of age were intraperitoneally administered 3% of thioglycollate. Four days after injection, peritoneal cells were harvested with ice-cold phosphate-buffered saline, washed, and suspended in Iscove's modified Dulbecco's medium (Life Technologies, Carlsbad, CA) containing 10% fetal calf serum. After removal of nonadherent cells, macrophages were incubated with the same medium in the presence of either dsFv anti-FRb or Ig V H -PE38 for 72 or 96 hours at 37°C in an atmosphere of 5% CO 2 . Propidium iodine (Sigma-Aldrich, Seelze, Germany) staining was performed as described previously. 12 Briefly, cells were stained with propidium iodine in 0.1% sodium citrate and 0.1% Triton X-100, and apoptotic cells were determined by a decrease in propidium iodine staining as detected by flow cytometry. Induced apoptosis (%) was determined by subtracting the percentage of apoptotic cells caused by IgV H -PE38 from that of dsFv anti-FRb in each sample. To analyze the FRb expression on thioglycollate-elicited peritoneal cells, adherent cells were stained with allophycocyanic-conjugated anti-FRb mAb, fluorescein isothiocyanate-F4/80 mAb (Biolegend, San Diego, CA), or allophycocyanic-or fluorescein isothiocyanate-conjugated isotype control mAb (Biolegend).

Quantitative Real-Time PCR
Quantitative real-time PCR (RT-PCR) with the TaqMan Probe method was performed to determine the differential expression of FRb and TNF-a mRNAs relative to the expression of glyceraldehydephosphatedehydrogenase (GAPDH) mRNA. Total RNA was extracted from the microdissected aorta by using the RiboPure Kit (Ambion, Austin, TX), followed by reverse transcription of the isolated RNA with the use of a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The first-strand cDNA was used as a template, and RT-PCR was performed with the use of the ABI PRISM 7300 Sequence Detection System (Applied Biosystems) with TaqMan Universal PCR Master Mix (Applied Biosystems). Primers and probes for FRb, TNF-a, and GAPDH were purchased from Applied Biosystems. Primer sequences for FRb were 5′-AA GGCTGACTCCCGTCTGTA-3′ (forward) and 5′-GCTCTTTACGC-CAACTCTGG-3′ (reverse), and the probe sequence was 5′-TAAGAGTCACTTCATCCAAG-3′.

Statistical Analysis
Data are presented as the standard error of the mean (SEM). For comparison of multiple independent groups, Kruskal-Wallis test followed by Holm's pairwise comparison was used. For the assessment of treatment effect and age relationships, data were analyzed with a 2-way factorial analysis of variance. A paired Student t test was used to compare the in vitro effect of immunotoxin and toxin. A probability value of P<0.05 was considered statistically significant.

FRb Expression in Atherosclerotic Lesions
We first evaluated the presence and localization of FRbexpressing cells in the aortic sinus lesion of the apoE-deficient mice at 21 weeks of age. We compared the distribution of Oil Red O staining to that of FRb-expressing cells in the serial slides (Figure 2A    FRb. Many of the FRb-expressing cells were observed in the intimal elastic lamina side of the intima, where macrophages tended to be larger than those of the luminal side. At 21 weeks of age, double fluorescence immunohistochemistry demonstrated colocalization of FRb with CD68 ( Figure 3A), but FRb was not colocalized with a-SMA, CD31, or CD3 ( Figure 3B through 3D). The percentages of FRb-expressing cells among the macrophage population were 57.2±2.9% at 21 weeks and 69.7±3.0% at 35 weeks of age.

FRb Immunotoxin Administration Attenuates Progression of Atherosclerosis
We used younger and older mice of 15 and 35 weeks of age in these studies. Each age group was divided into immunotoxin, toxin, and control groups and received the dsFv anti-FRb-PE38 immunotoxin, PE38, or saline, respectively, every 3 days for a total of 5 times. When the mice were euthanized at 21 and 41 weeks of age, the immunotoxin group showed a 31% and 22% reduction in atherosclerotic lesions, respectively ( Figure 4A and 4B), compared with the control and toxin groups (21 weeks: control=20.9±1.7%, immunotoxin=14.4±1.4%, toxin=20.8±1.5%, P=0.03, n=8; 41 weeks:control=36.2±1.5%, immunotoxin=28.1±5.6%, toxin= 35.7±1.3%, P=0.02, n=5) ( Figure 4C and 4D). Treatment with PE38 had no effect on the size of atherosclerotic lesions, and there was no interaction between age and the effect of the treatments. No significant differences were observed among the 3 groups in body weights, serum levels of total cholesterol, or numbers of circulating neutrophils, eosinophils, monocytes, and lymphocytes in peripheral blood. We also used 2-factor analysis of variance for several measurements that might affect the differences by treatment in the 2 age groups, such as body weights, total cholesterol levels, the number of monocytes, and the number of eosinophils. Of these data, there were significant differences in the number of eosinophils and monocytes between the 2 age groups. However, immunotoxin or toxin had no effect on the numbers of eosinophils or monocytes, and there was no interaction between age and the drug effect. (Table 1).

Immunohistochemical Analysis of Atherosclerotic Lesions After FRb Immunotoxin Administration
Administration of FRb immunotoxin significantly reduced the percentage of CD68-, FRb-, and TNF-a-stained area compared with the toxin or control group at both 21 and 41 weeks of age ( Figure 5A and 5B). There was a slight decrease in the percentage of FRb-expressing cells in the toxin group compared to the control group (P=0.04) at 41 weeks of age only. This observation could indicate the possibility of a nonspecific effect of the toxin on phagocytic cells. 22 Although a-SMA-expressing cells tended to decrease in the immunotoxin groups at 21 and 41 weeks of age, these differences did not reach statistical significance, and there was no significant difference in CD3-expressing cells ( Table 2).

Effects of FRb Immunotoxin on Cell Proliferation and Apoptosis in Atherosclerotic Lesions
Because the effect of immunotoxin on apoptosis wears off within a few days and apoptosis cells induced by immunotoxin were not detected 4 weeks after the last injection (probably because of the removal of apoptotic cells), PCNA and TUNEL staining was performed 1 week after the last injection to assess the level of cell proliferation and apoptosis ( Figure 6). As shown in Table 2, the percentage of apoptotic cells in the immunotoxin group was significantly higher than that in the control and toxin groups. Also, the percentage of cells expressing PCNA was significantly decreased compared with the control and toxin groups in both age groups.

In Vitro Effect of FRb Immunotoxin
We stained thioglycollate-elicited peritoneal cells by using anti-FRb mAb and anti-F4/80 mAb, and the percentage of peritoneal macrophages expressing FRb was 95.4±1.3%

RT-PCR Analysis of mRNA Expression of FRb and TNF-a in Atherosclerosis After FRb Immunotoxin Administration
We

Discussion
This study is the first demonstration that FRb-expressing macrophages are present in the atherosclerotic lesions and that intravenous administration of FRb immunotoxin markedly reduced the progression of atherosclerotic lesions in both the early and advanced stages of atherosclerosis. It has been reported previously that FRb is expressed on activated macrophages at the site of inflammation. 11 Previous work that used a polyclonal antibody to FRb suggested that FRb was a neutrophilic lineage marker. 23 However, we used a mAb specific to FRb and reported that FRb was not expressed by peripheral blood neutrophils, even after in vitro stimulation with phorbol myristate acetate. 12 In the present study, we demonstrated significant coexpression of FRb with macrophages, but not with endothelial cells, vascular smooth muscle cells, or T cells. Moreover, histological analysis revealed that FRb immunotoxin treatment resulted in a significant reduction of macrophages expressing CD68, FRb, and TNF-a. In addition, the administration of FRb immunotoxin also increased the percentage of TUNEL-positive apoptotic cells and reduced the percentage of PCNA-positive proliferating cells. RT-PCR performed on aorta demonstrated reduced mRNA expression of FRb and TNF-a in mice treated with immunotoxin, which is in accordance with the histological data. Macrophages play an important role in atherosclerosis by promoting inflammatory responses. Although targeting macrophages in atherosclerotic lesions seems to be a promising strategy for preventing the progression of atherosclerosis, the antiatherogenic effect of killing macrophages within the plaques remains controversial. 6,[24][25][26][27] The difference in the phagocytic clearance of dead macrophages might lead to conflicting results according to the lesion stage. In the early stage of atherosclerosis, induction of macrophage apoptosis is beneficial because the phagocytic clearance of apoptotic macrophages is efficient during this stage. 24 In contrast, in the advanced stage of atherosclerosis, increasing macrophage apoptosis is detrimental because of the defective phagocytic clearance of accumulated dead macrophages. 26,27 Induction of macrophage apoptosis in the advanced stage could lead to the progression of atherosclerotic lesions, but previous studies targeted pan-macrophage. We targeted a selected subset of macrophages that engage inflammation and demonstrated that the dsFv anti-FRb immunotoxin caused apoptosis in the thioglycollate-elicited peritoneal macrophages. In addition, administration of dsFv anti-FRb immunotoxin increased apoptosis and suppressed the progression of atherosclerotic lesions in both the early and advanced stages of atherosclerosis. The majority of apoptotic cells in atherosclerotic lesions are macrophages, 4 and cellular proliferation within atheroma involves predominantly vascular smooth muscle cells and macrophages. 28,29 By induction of apoptosis in FRb-expressing macrophages, growth factors and chemokines, which stimulate the proliferation of macrophages and smooth muscle cells, might decrease. Although there was not a significant difference in the number of smooth muscle cells between the control, immunotoxin, and toxin groups, the number of smooth muscle cells tended to decrease in the immunotoxin group. Taken together, elimination of a selected subset of macrophages that engage actively in inflammation might leave the clearance of apoptotic cells undisturbed, lead to the suppression of cell proliferation, and thus, exert favorable suppressive effects on the progression of atherosclerotic lesions in both the early and late stages of atherosclerosis.
In the immunohistochemical study, FRb-expressing macrophages were observed mainly on the medial side of the intima, where macrophages tended to be larger than those on the luminal side. Although most of the FRb-expressing cells were CD68 positive, some of them were CD68 negative. These FRb + /CD68 À cells were noted to be anucleate and exhibited patchy deposits of Oil Red O staining, and they thus could represent the cellular remnants of dead macrophages. CD68 is expressed strongly in cytoplasmic granules of cells of the monocyte/macrophage lineage, whereas FRb is expressed on the cell membrane. The difference in staining pattern between CD68 and FRb should be able to explain the presence of CD68 À /FRb + cells observed at anucleate area.
FRb-expressing macrophages were reported to have produced TNF-a and reactive oxygen species 14 and to act as M1 macrophages in rheumatoid arthritis. 30 However, it remains controversial whether FRb expression reflects M1 function because FRb macrophages are considered to be M2 macrophages in glioma 16 and melanoma. 31 The patterns of gene expression of M1/M2 macrophages vary in response to various environmental stimuli, and FRb in atherosclerotic lesions is heterogeneous and does not precisely fit either M1 or M2 in vivo. Although phenotypic variation among activated macrophages has not yet been completely validated, FRb-expressing macrophages expressing TNF-a are often noted to be in lipid-laden macrophages, which eventually develop into foam cells. Targeting these FRb-expressing macrophages might be an effective strategy to suppress inflammation in atherosclerotic lesions.
Several immunotoxins consisting of mAbs conjugated to toxins have been used for clinical trials, such as in acute lymphoblastic leukemia, 32 hairy cell leukemia, 33 and mesothelioma. 34 The truncated Pseudomonas aeruginosa exotoxin known as PE38 lacks its binding domain. 35 Our recombinant dsFv anti-FRb-PE38 immunotoxin consisted of the truncated PE38 and the heavy-and light-chain portions of the anti-FRb antibody. When FRb-expressing macrophages internalize the immunotoxin, the PE fragments travel through the endosomes into the trans-Golgi network. The PE fragments then are secreted into the cytosol, where they inhibit protein synthesis by elongation factor-2, leading to apoptosis. 36,37 Because FRb is expressed exclusively on activated macrophages in atherosclerotic lesions, FRb immunotoxin could be capable of mediating macrophage apoptosis in patients with ischemic heart diseases and peripheral arterial diseases thorough its specificity and cytotoxicity. Several problems such as nonspecific toxicity 32,38 and immunogenicity 39 should be solved before the clinical use of the immunotoxins can be adopted. Although elevation of hepatic transaminases was observed as an adverse effect of nonspecific toxicity, this proved to be reversible and had an acceptable safety profile. 32 In addition, the humanized immunotoxins, with their cytotoxic moiety based on proteins or RNase of human origin, 40-42 might overcome problems with immunogenicity.
Several attempts have been made to inhibit the development of atherosclerotic lesions by administering antibodies against mediators such as placental growth factor, CD40L, macrophage colony-stimulating factor, and monocyte chemoattractant proteins 1 and 5. [43][44][45][46] These interventions have induced regression of atherosclerotic lesions by blocking certain epitopes or chemokines on macrophages and vascular smooth muscle cells, resulting in reduced macrophage recruitment and the downregulation of proinflammatory pathways. In contrast to those studies, we used FRb immunotoxin to induce apoptosis of activated macrophages rather than targeting individual chemokines. The elimination of activated FRb-expressing macrophages could prevent activated macrophages from releasing inflammatory cytokines and recruiting circulating macrophages and thus could break the vicious circle operating in the atherogenic environment.
Previous reports have indicated that folate conjugates detect sites of inflammation in adjuvant-induced arthritis and atherosclerosis, which involve activated macrophages. 15,47,48 Although the uptake of folate conjugates was mediated by the FR, it is possible that other FR isoforms were involved. In the present study, we targeted the b isoform of FR that is only expressed by activated macrophages. We believe that the cell specificity of this method might be of particular value in delivering imaging or therapeutic agents to atherosclerotic lesions. We found that FRb was expressed by macrophages in the atherosclerotic lesions of human carotid and renal arteries (data not shown). The FRb antibody could be used as an imaging agent to distinguish vulnerable plaques in human atherosclerosis that are prone to rupture, resulting in myocardial infarction.
In conclusion, we have demonstrated the presence and distribution of FRb-expressing macrophages in atherosclerotic lesions. A recombinant FRb immunotoxin reduced FRb and TNF-a mRNA expression and the number of FRb-and TNF-a-expressing macrophages and thus suppressed the progression of atherosclerotic lesions. Targeting activated macrophages with the FRb immunotoxin could be beneficial in the suppression of both the early and advanced stages of atherosclerosis. Although further clinical research would be required to confirm the effect of killing activated macrophages in human atherosclerotic lesions, our findings provide a new strategy for targeting activated macrophages, with clinical implications for the treatment of atherosclerosis.