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mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody a
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Yosdel Soto , Niurka Mesa , Yumisley Alfonso , Arlenis Pérez , Fernando Batlle , Tania a
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Griñán , Adonis Pino , Justo Viera , Milagros Frómeta , Victor Brito , Armando Olivera , b
Francisco Zayas & Ana M Vázquez a
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Research and Development Direction, Center of Molecular Immunology, Havana, Cuba
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Department of Nuclear Medicine, Institute of Nephrology, Havana, Cuba
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Parasitology Department, Institute of Tropical Medicine «Pedro Kourí», Havana, Cuba
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Experimental Surgery Department, Center of Medical and Surgical Research, Havana, Cuba Published online: 30 Oct 2014.
To cite this article: Yosdel Soto, Niurka Mesa, Yumisley Alfonso, Arlenis Pérez, Fernando Batlle, Tania Griñán, Adonis Pino, Justo Viera, Milagros Frómeta, Victor Brito, Armando Olivera, Francisco Zayas & Ana M Vázquez (2014) Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody, mAbs, 6:5, 1340-1346, DOI: 10.4161/mabs.29970 To link to this article: http://dx.doi.org/10.4161/mabs.29970
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REPORT mAbs 6:5, 1340--1346; September/October 2014; © 2014 Taylor & Francis Group, LLC
Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody ~an1, Adonis Pino3, Justo Viera1, Yosdel Soto1, Niurka Mesa2, Yumisley Alfonso2, Arlenis Perez1, Fernando Batlle2, Tania Grin 1 1 4 2 meta , Victor Brito , Armando Olivera , Francisco Zayas , and Ana M Vazquez1,* Milagros Fro 1
Research and Development Direction, Center of Molecular Immunology, Havana, Cuba; 2Department of Nuclear Medicine, Institute of Nephrology, Havana, Cuba; 3Parasitology Department, Institute of Tropical Medicine «Pedro Kourí», Havana, Cuba; 4Experimental Surgery Department, Center of Medical and Surgical Research, Havana, Cuba
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Keywords: monoclonal antibodies, glycosaminoglycans, atherosclerosis, technetium-99m, imaging Abbreviations: % ID/g, percentage of injected dose per gram of tissue; At-R, Atherosclerotic rabbits; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycans; DS, dermatan sulfate; ELISA, enzyme-linked immunoadsorbent assay; GAG, glycosaminoglycan; LDL, low density lipoprotein; mAb, monoclonal antibody; Non At-R, Non atherosclerotic rabbit; NZW rabbits, New Zealand White rabbits; PG, proteoglycans
The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n D 14) by the administration of Lipofundin 20% using PBSreceiving animals as control (n D 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of 99mTc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of 99mTc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, 99mTc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of 99mTc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.
Introduction Atherosclerosis is the underlying pathology of most cardiovascular events and the main cause of morbidity and mortality worldwide.1 This disease is characterized by a non-resolved inflammatory response triggered by the retention of apoB-containing lipoproteins in the artery wall.2,3 Upon entrapment in the intima layer, proatherogenic lipoproteins undergo oxidative and enzymatic modifications thereby increasing their immunogenicity.4 These modifications also trigger the uptake of lipoprotein by macrophages and vascular smooth muscle cells, which leads to foam cells formation.4,5 Subendothelial retention of low density lipoprotein (LDL) is mediated by electrostatic interactions between positively charged residues on apolipoprotein B-100 (apoB-100) and negatively
charged glycosaminoglycans (GAGs) chains on proteoglycans (PGs).6,7 A prominent feature of atherosclerosis progression is the accumulation of chondroitin sulfate (CS) and dermatan sulfate (DS) PGs, accompanied by hyperelongation of GAGs side chains.8,9 CSPG-LDL complexes have been detected in injured rabbit intima,10 and they have also been isolated from human atherosclerotic lesions.11 In a previous work, we characterized the antiatherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99, which binds sulfated GAGs, inhibits LDL binding to CS, and abrogates LDL oxidation in vitro. Moreover, rabbits immunized with chP3R99 mAb showed reduced aortic arch lesions and no macrophage infiltration following short-term treatment with Lipofundin 20% (referred to as Lipofundin).12 Similar results were observed in ApoE¡/¡ mice fed with a
*Correspondence to: Ana M Vazquez; Email: [email protected] Submitted: 04/01/2014; Revised: 07/09/2014; Accepted: 07/16/2014 http://dx.doi.org/10.4161/mabs.29970
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Figure 1. chP3R99 mAb reactivity to Lipofundin-induced atherosclerotic plaque from rabbit aorta. GAGs were detected in the artery wall of normal and Lipofundin-treated rabbits by Alcian Blue staining and immunohistochemistry with CS-56 mAb (1 mg/mL). Samples from normal aorta and sections corresponding to areas with early and advanced lesions were incubated with chP3R99 mAb or the isotype-matched control hR3 (2 mg/mL). Original magnification X20. Scale bars D 100 mm.
high-fat high-cholesterol diet in which the administration of chP3R99 mAb reduced the total lesion area in more than 40%.13 In this paper, we demonstrated that chP3R99 mAb preferentially accumulates in rabbit atherosclerotic lesions, supporting the use of anti-GAGs antibodies for the detection and treatment of atherosclerosis.
Results chP3R99 mAb reactivity to rabbit atherosclerotic lesions To evaluate the content of GAG in the artery wall, aortic sections from normal and Lipofundin-treated rabbits were stained with Alcian Blue. As depicted in Figure 1, the amounts of GAGs increased in the aortic arch depending on the presence and size of the lesions. Particularly, immunohistochemistry revealed a high content of CS in the aortic lesions of Lipofundin-receiving rabbits, which results in a strong reactivity of chP3R99 mAb in these areas. A lower reactivity was observed in aortic sections from the control rabbits and in Type I AHA lesions, whereas the isotypematched control (hR3 mAb) did not react with any of the aortic sections.
intensity of the fluorescence were lower in the aorta of healthy animals, and located predominantly in the adventitia (Fig. 2D–F). In fact, as depicted in Figure 2J, the accumulation of chP3R99 was significantly higher (8.7-fold) in atherosclerotic rabbits than in controls; and also higher (135-fold) than the accumulation of the isotype-matched mAb in atherosclerotic rabbits (P < 0.05). These results were confirmed by immunoscintigraphy by intravenous administration of 99mTc-chP3R99 mAb in atherosclerotic and control rabbits (Fig. 3). Ten minutes after the injection of 99mTc-chP3R99 mAb or the isotype-matched control 99m Tc-chT3 mAb, blood-pool images were similar in all animals (data not shown). The overall uptake was predominantly localized in liver, kidneys and heart alongside all time intervals. Planar images acquisition revealed the accumulation of 99mTc-chP3R99 mAb in the carotid of atherosclerotic rabbits 6 h after radiotracer administration (Fig. 3A), but not in control animals (Fig. 3B). The visualization of atherosclerotic lesions upon 99mTc-chP3R99 mAb injection was specific, since no evident accumulation of 99m Tc-chT3 mAb was observed in Lipofundin-receiving rabbits (Fig. 3C). 99m
chP3R99 mAb accumulation in Lipofundin-induced lesions The accumulation of chP3R99 mAb in atherosclerotic lesions in vivo was addressed by immunofluorescence upon intravenous injection of 1 mg of the mAb in rabbits receiving or not Lipofundin (Fig. 2). Unlike hR3 mAb (Fig. 2A–C), chP3R99 was detected in lesioned arterial tissue, adventitia and the surface of smooth muscle cells of atherosclerotic rabbits (Fig. 2G–I). In contrast, both the presence of this anti-GAGs mAb and the
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Tc-chP3R99 mAb arterial uptake The distribution of 99mTc-chP3R99 mAb in rabbits is summarized in Figure 4. The percentage of injected dose of the radiotracer per gram of tissue (% ID/g) in samples of Lipofundin-treated rabbits was greater in kidney (21.3 § 1.8% ID/g) and urine (15.7 § 6.0% ID/g). As depicted in Figure 4A and 4B, we found similar mAb uptake by non-targeted organs without marked differentiation between lesioned and non-lesioned rabbits, (P > 0.05). In contrast, 99m Tc-chP3R99 mAb accumulation into atherosclerotic lesions was
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greater than the one observed in the artery wall of control rabbits, both for aortic arch (1.019 § 0.294% ID/g vs. 0.187 § 0.097% ID/g) and thoracic segment (0.547 § 0.180% ID/g vs. 0.097 § 0.035% ID/g), (P < 0.05). In these segments, the accumulation of radiolabeled mAb was more than 5-fold higher in Lipofundin-receiving rabbits than in controls (Fig. 4B). According to immunoscintigraphy images, we 99m Figure 2. Immunofluorescence detection of in vivo chP3R99 accumulation in Lipofundin-induced atherosclerotic found that Tclesions. Representative images from atherosclerotic rabbits injected with 1 mg of the isotype matched control (A–C) chP3R99 mAb uptake by or chP3R99 mAb (G–I). Frozen sections from healthy rabbits who received the same mass of chP3R99 are shown (D–F). carotids with lesions was Human antibodies were detected through the incubation with a goat phycoerythrine (PE)-conjugated anti-human IgG 3.9-fold higher than that followed by counterstaining with Hoechst. Original magnification X20. Scale bars D 100 mm. (J) Represents the percentages of human IgG stained areas respect to total area. At-R D atherosclerotic rabbits; NAt-R D non atherosclerotic rabin controls (0.597 § bits. Data are expressed as mean § SD. 0.079% ID/g vs. 0.157 § 0.140% ID/g), (P < 0.05). Although the % ID/g in abdominal portion of aorta from Discussion Lipofundin-receiving animals was 2.8-fold higher than in non-athThe use of therapeutic agents targeting the arterial wall, espeerosclerotic rabbits, no significant differences were observed between these groups (0.356 § 0.174 ID/g vs. 0.139 § 0.121% cially those aimed at preventing the retention of proatherogenic ID/g), (P > 0.05). Also, chP3R99 mAb accumulation in aortic arch from rabbits with atheromatous plaques was 1.7-fold and 2.5-fold higher than in thoracic and abdominal segments, respectively. In addition, the % ID/g ratio of arterial segments-to-muscle and arterial segments-toblood were significantly greater in Lipofundin-receiving rabbits than in controls (P < 0.05) (Table 1). Histopathological study 99m Tc-chP3R99 mAb accumulation in different segments of the aorta and carotids was associated to lesions size of Lipofundinreceiving rabbits (Fig. 5). Histopathological studies revealed the presence of atherosclerotic lesions in aortic arch (Fig. 5A and 5B) and thoracic segment (Fig. 5C) of aorta and carotids (Fig. 5D), characterized by intimal thickening, leukocyte infiltration and distortion of smooth muscle cells arrangement. In contrast, only a slight thickening of the intima was observed in the abdominal segment (Fig. 5E). The aortas from PBS-receiving rabbits showed no alteration in the vascular architecture (Fig. 5F).
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Figure 3. Immunoscintigrams of rabbits injected with 99mTc-chP3R99 mAb or 99mTc-chT3 mAb. Planar images acquired at 6 h after radiotracers injection, showed a selective accumulation of chP3R99 mAb in carotids (arrowhead) from rabbits with Lipofundin-induced atherosclerotic lesions (A) but not in healthy rabbits (B). No accumulation of the isotype-matched control mAb was observed in atherosclerotic lesions (C).
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reduce lesions size or to halt their growth. In rabbits, the acute administration of Lipofundin induces American Heart Association type IIIIV atherosclerotic lesions characterized by accumulation of abundant GAGs and collagen fibers in the subendothelial space, along with macrophages infiltration.12 According to the in vitro results, immunoscintigraphy and immunofluorescence analysis revealed a preferential accumulation of chP3R99 mAb in atherosclerotic plaques in rabbits. In Figure 4. Biodistribution of 99mTc-chP3R99 mAb in rabbits determined as % ID/g 6 h after radiotracer administracontrast to the isotypetion. (A) Radiotracer uptake by organs. Black and white bars represent the % ID/g in atherosclerotic and healthy matched control, chP3R99 rabbits, respectively. (B) Ratio of 99mTc-chP3R99 mAb uptake by samples from rabbits with Lipofundin-induced athmAb showed sustained accuerosclerosis to non-atherosclerotic rabbits. Data are expressed as mean § SD. At-R D atherosclerotic rabbits; NAt-R mulation within carotids D non atherosclerotic rabbits. from Lipofundin-treated rabbits, but not in the arterlipoproteins in the subendothelial matrix, is a promising strategy ies of control animals, resulting in a specific uptake by atherosclewhich has been poorly addressed.14 Previously, we reported that rotic lesions. These results agreed with a higher detection of the chP3R99 mAb prevents atherosclerosis in rabbits; presumably chimeric antibody by immunofluorescence in frozen aortic secthrough the induction of autologous anti-CS antibodies gener- tions from rabbits with lesions. In the same line, Brito and cowated upon immunization. However, a direct proof of the reactiv- orkers reported that chP3R99-fluorescein isothiocyanate injected ity of chP3R99 mAb or those antibodies with the rabbit artery intravenously colocalizes with activated endothelial areas of aorta wall was not demonstrated.12 Now, we showed that chP3R99 in ApoE¡/¡ mice fed a high-fat high-cholesterol diet.13 mAb specifically recognizes atherosclerotic lesions, in a close assoDue to the small size of atherosclerotic plaques and the close ciation with their size and the presence of CS GAGs. The devel- proximity of aorta to heart and liver we could not visualize this opment of atherosclerosis is characterized by a shifting of artery by immunoscintigraphy. Notwithstanding, as revealed in contractile vascular smooth muscle cells toward secretory pheno- biodistribution studies, the uptake of 99mTc-chP3R99 mAb by type, thereby affecting the composition and atherogenicity of aortic arch and thoracic segment from atherosclerotic rabbits was PGs produced.15 For instance, it have been described a promi- almost 6-folds higher than by the arterial wall of healthy animals. nent accumulation of CSPGs, throughout the artery wall16 with In addition, radiotracer uptake was not evenly distributed elongated GAGs chains and higher sulfation degree at atheroscle- throughout the descending aorta and was related with the size of lesions detected in these areas. Indeed, 99mTc-chP3R99 accumurosis-prone sites.17,18 Hence, these results might suggest the feasibility of using this lation in the aortic cross was 2.5-fold greater than in abdominal anti-GAG mAb in advanced stages of the disease in order to segment in atherosclerotic rabbits. Although it has been described that LDL retention in the artery wall is similar among all aortic sites of normal rabbits, it is increased at atherosclerosis-susceptible areas as soon as 4 d after feeding with hypercholesterolemic Table 1. Biodistribution of chP3R99 mAb in arterial segments diet.19 Control rabbits Atherosclerotic rabbits Here, we also demonstrated that Lipofundin, in addition to (n D 3) (n D 4) develop atherosclerosis in aorta, also induces lesions in carotids as Arterial Segment A/M A/B A/M A/B reflected by the thickening of the intima layer, extracellular matrix production and distortion in the arrangement of smooth Aortic Arch 0.47 § 0.26 0.14 § 0.12 7.09 § 1.12* 0.93 § 0.36* 99m TcThoracic 0.80 § 0.30 0.09 § 0.07 5.12 § 1.59* 0.50 § 0.32* muscle cells. Thus, we found almost a 4-folds increase in chP3R99 mAb uptake into this artery, supporting the accumulaAbdominal 0.55 § 0.09 0.16 § 0.12 2.34 § 0.08* 0.55 § 0.38 Carotids 0.77 § 0.19 0.27 § 0.18 2.67 § 0.35* 0.75 § 0.19* tion of the mAb observed by immunoscintigraphy. Until now, most of the efforts in the development of radioValues are expressed as mean § SD. A/M: arteria-to-muscle ratio; tracers to detect atherosclerotic lesions have been focused on the A/B: arteria-to-blood ratio; *P < 0.05 vs. control group. identification of important pathways associated with plaque
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instability, such as macrophage recruitment, matrix breakdown enzyme, macrophage metabolism, thrombosis and apoptosis.20,21 However, since CS/DS PGs are produced in large quantities during all the stages of human atherosclerosis development they could be a good target to detect atherosclerotic lesions, even in early phases of the disease.22,23 This could be useful in atherosclerosis diagnosing as a criterion to prescribe artery wall-based therapies.17 Because we used as radiotracer a full-sized mAb tagged with 99Tc, there was a slow blood clearance of the probe, resulting in a suboptimal target-to-background ratio. Hence, the 99m Tc-chP3R99 mAb uptake detected by immunoscintigraphy in the lesioned carotids could be attributable to both specific accumulation and blood pool radiactivity. However, it was possible to observe a focal accumulation of chP3R99 mAb in some regions of carotids from Lipofundin-receiving rabbits along with higher A/B and A/M ratios in these animals. Conversely, we could not detect neither chP3R99 mAb in carotids from healthy animals nor accumulation of the isotype-matched control mAb in atherosclerotic plaques. Future studies using fragments of chP3R99 mAb (Fab, scFv) or engineered variants (diabodies, triabodies, minibodies, and single-domain antibodies) are required Figure 5. Representative atherosclerotic lesions developed in aortic segments and carotids of in order to optimize the targeting of atheroLipofundin-receiving rabbits. Hematoxylin-Eosin shows intimal thickening, leukocyte infiltration sclerotic lesions with a fast clearance from and distortion in the vascular tissue architecture in aortic arch (A, B), thoracic segment (C) and circulation.24,25 A recent approach for carcarotids (D) of Lipofundin-treated rabbits. Minor lesions were observed in the abdominal segment of aorta (E) whereas no alteration in the artery wall of normal rabbits was detected (F). diovascular applications is the use of nanoOriginal magnification X20 except for B (X40). Scale bars D 50 mm except for (D) where bodies as radiotracers, displaying high bar D 100 mm. stability, fast blood clearance, and the possibility of labeling with different probes thereby allowing atherosclerotic lesions imaging.26 of Laboratory Animals (CENPALAB, Mayabeque, Cuba). AniIn summary, our results show that chP3R99 mAb preferen- mals were housed under standard conditions (25 C, 60 § 10% tially accumulates in atherosclerotic lesions, even at early phases humidity), 12 h day/night cycles with water and food ad libitum. of the disease, supporting the potential use of this anti-GAGs All procedures were approved by the Institutional Animal Ethic antibody for diagnosing and treatment of atherosclerosis. Committee.
Materials and Methods Antibodies chP3R99, chT3 (anti-human CD3)27 and hR3 (anti-Epidermal Growth Factor receptor)28 mAbs were purified by protein-A affinity chromatography (Pharmacia) from transfectoma culture supernatants and analyzed by SDS-PAGE. Animals New Zealand White (NZW) male rabbits (3.0–3.5 kg, 12 wk old) were obtained from the National Center for the Production
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Radiolabeling of mAbs Labeling of mAbs with 99mTc was performed according to a method previously reported.29 Briefly, mAbs were reduced at a 2-mercaptoethanol-to-mAb molar ratio of 850:1 at room temperature (RT) for 30 min. After purification by a PD-10 column chromatography (Sephadex G-25), the reactivity of the reduced mAbs to CS (Sigma) was determined by ELISA, as previously described.12 Then, 84 mg of sodium pyrophosphate and 2 mg of stannous chloride were diluted in 5.0 mL of sodium chloride 0.9% and 100 mL of this mixture were added to 1 mg of the reduced mAbs. The samples were incubated for 15 min at RT followed by a 30 min incubation with 1295 MBq (35 mCi)
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99m
Tc pertechnetate eluted from a 99mMo–99mTc generator (CENTIS, Mayabeque, Cuba). Quality control was determined by thin-layer chromatography using both acetone and sodium chloride 0.9% as mobile phase yielding >90% of radiochemical purity.
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Cysteine and rabbit sera challenge Transchelation experiments were performed by challenging 31 mg of 99mTc-chP3R99 mAb with 630 nmol of cysteine at a final molar ratio cysteine to mAb 3000:1, and rabbit sera (volume ratio 20:1) at RT for 24 h. In these extreme conditions we found 40–50% of displacement of the radioactivity. Accumulation of chP3R99 mAb in atherosclerotic lesions Atherosclerosis was induced in 18 NZW rabbits by daily administration of Lipofundin 20% (Braun Melsungen) (2 mL/ kg, 8 d) through marginal ear vein, as previously described.12,30 Eight rabbits that received only PBS were used as control. To evaluate the preferential accumulation of chP3R99 mAb in atherosclerotic lesions, six Lipofundin-treated rabbits and four healthy controls intravenously received 1mg of this antibody into the marginal ear vein the day after the last dose of Lipofundin and were sacrificed 24 h later. Specificity was assessed by administrating the same mass of hR3 mAb in four atherosclerotic rabbits. In other set of experiments, 99mTc-chP3R99 mAb (0.3 mg, 370 MBq) was injected into the marginal ear vein both in Lipofundin-treated (n D 4) and PBS-receiving rabbits (n D 3). As a control of specificity, two atherosclerotic rabbits received the same dose of 99mTc-chT3 mAb. After 12 h of fasting, rabbits were anesthetized with 35 mg/kg of ketamine hydrochloride and 5 mg/kg of xylazine HCl, IM. Radioimmunoscintigraphy was performed 10 min after radiotracers injection and repeated 6 h later. Planar images were obtained in the anterior views with a gamma camera (MEDISO) equipped with a low-energy, parallelhole, high-resolution collimator and stored on a 128 £ 128 pixel matrix by means of a digital computer. Images were recorded until 200K counts at the energy peak of 140 keV with a 20% window. Rabbits were sacrificed by intracoronary injection of KCl under anesthesia and the following organs and tissues were removed, weighed, and counted in a well scintillation g-counter (CAPINTEC, Inc. CRC-25W): aorta (aortic arch, thoracic and abdominal segments), carotids, heart, lung, liver, spleen, kidney and muscle. The radioactivity of blood and urine was counted as well. Radioactivity uptake was expressed as percentage of injected dose per gram of tissue (% ID/g). Specific antibody accumulation in organs was considered when the ratio of % ID/g of atherosclerotic-to-control rabbits was >2.5. References 1. Negi S, Anand A. Atherosclerotic coronary heart disease-epidemiology, classification and management. Cardiovasc Hematol Disord Drug Targets 2010; 10:257-61; PMID:20932265; http://dx.doi.org/ 10.2174/187152910793743832 2. Tabas I, Glass CK. Anti-inflammatory therapy in chronic disease: challenges and opportunities. Science 2013;
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Histological study Histopathological findings in the different segments of aorta and carotids as well as immunohistochemical studies were performed in formalin-fixed (24 h, pH 7.4) paraffin-embedded tissue samples. The specimens were cut into 5-mm slices, deparaffinized, rehydrated, and stained with hematoxylin-eosin or Alcian Blue for light microscopic examination. The reactivity to the artery wall was evaluated in aortic sections from two rabbits in which the atherosclerotic lesions were induced or unmanipulated ones. After blocking endogenous peroxidase activity (Dako), sections were incubated with chP3R99 mAb or the isotype-matched control hR3 (2 mg/mL) for 1 h at RT, followed by an anti-human IgG serum conjugated to peroxidase (Jackson ImmunoResearch Lab). Anti-CS mAb (clone CS-56, Sigma) was used as positive control (1 mg/mL) and it was identified by the incubation with the EnVisionTM Detection Systems (Dako). Finally, sections were incubated with diaminobenzidine substrate solution and counterstained with hematoxylin. The presence of chP3R99 mAb in the artery wall was detected by immunofluorescence in fresh frozen samples, by incubating tissue sections with a goat phycoerythrine (PE)-conjugated antihuman IgG (BD Bioscience), followed by counterstaining with Hoechst. Images were digitally captured with a DP20 camera coupled to a fluorescence microscope Olympus BX51. Fluorescence images were quantified by using the software ImageJ 1.47. Statistical analysis Data are expressed as the mean § SD. To compare the accumulation of the chimeric antibodies in the artery wall of rabbits in immunofluorescence experiments a one-way ANOVA was performed followed by Bonferroni post hoc test. The % ID/g in organs and ratios between atherosclerotic rabbits and controls were compared by use of the Student t test. A P < 0.05 value was considered statistically significant.
Disclosure of Potential Conflicts of Interest
Ana Marıa Vazquez, Yosdel Soto and Victor Brito are inventors of a patent related with antibodies that recognize sulfatides and sulfated PGs; however, they have assigned their rights to the assignee Center of Molecular Immunology.
Acknowledgments
This work was supported by the Center of Molecular Immunology (Havana, Cuba).
339:166-72; PMID:23307734; http://dx.doi.org/ 10.1126/science.1230720 3. Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 2013; 339:161-6; PMID:23307733; http://dx. doi.org/10.1126/science.1230719 4. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol
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2006; 6:508-19; PMID:16778830; http://dx.doi.org/ 10.1038/nri1882 5. Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation 2007; 116:1832-44; PMID:17938300; http://dx.doi.org/ 10.1161/CIRCULATIONAHA.106.676890 6. Hurt-Camejo E, Olsson U, Wiklund O, Bondjers G, Camejo G. Cellular consequences of the association of
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7.
8.
Downloaded by [University of Western Ontario] at 02:33 05 February 2015
9.
10.
11.
12.
13.
apoB lipoproteins with proteoglycans. Potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol 1997; 17:1011-7; PMID:9194748; http://dx.doi.org/ 10.1161/01.ATV.17.6.1011 Khalil MF, Wagner WD, Goldberg IJ. Molecular interactions leading to lipoprotein retention and the initiation of atherosclerosis. Arterioscler Thromb Vasc Biol 2004; 24:2211-8; PMID:15472124; http://dx.doi.org/ 10.1161/01.ATV.0000147163.54024.70 Little PJ, Osman N, O’Brien KD. Hyperelongated biglycan: the surreptitious initiator of atherosclerosis. Curr Opin Lipidol 2008; 19:448-54; PMID:18769225; http:// dx.doi.org/10.1097/MOL.0b013e32830dd7c4 Ballinger ML, Osman N, Hashimura K, de Haan JB, Jandeleit-Dahm K, Allen T, Tannock LR, Rutledge JC, Little PJ. Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo. J Cell Mol Med 2010; 14(6B):1408-18; PMID:19754668; http://dx. doi.org/10.1111/j.1582-4934.2009.00902.x Srinivasan SR, Vijayagopal P, Dalferes ERJ Jr., Abbate B, Radhakrishnamurthy B, Berenson GS. Dynamics of lipoprotein-glycosaminoglycan interactions in the atherosclerotic rabbit aorta in vivo. Biochim Biophys Acta 1984; 793:157-68; PMID:6712964; http://dx.doi.org/ 10.1016/0005-2760(84)90317-5 Camejo G, Hurt E, Romano M. Properties of lipoprotein complexes isolated by affinity chromatography from human aorta. Biomed Biochim Acta 1985; 44:389-401; PMID:4004839 Soto Y, Acosta E, Delgado L, Perez A, Falcon V, Becquer Brito V, Alvarez MA, Fraga A, I, Gri~ nan T, et al. Antiatherosclerotic effect of an antibody that binds to extracellular matrix glycosaminoglycans. Arterioscler Thromb Vasc Biol 2012; 32:595-604; PMID:22267481; http://dx.doi. org/10.1161/ATVBAHA.111.238659 Brito V, Mellal K, Giroux S, Perez A, Soto Y, deBlois D, Ong H, Marleau S, Vazquez AM. Induction of anti-antiidiotype antibodies against sulfated glycosaminoglycans reduces atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2012; 32:2847-54; PM ID:2 3087361; http:/ /dx.doi.org/10 .1161/ ATVBAHA.112.300444
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14. Tabas I. Making things stick in the fight against atherosclerosis. Circ Res 2013; 112:1094-6; PMID:23580771; http://dx.doi.org/10.1161/CIRCRESAHA.113.301227 15. Manderson JA, Mosse PRL, Safstrom JA, Young SB, Campbell GR. Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype. Arteriosclerosis 1989; 9:289-98; PMID:2719591; http:// dx.doi.org/10.1161/01.ATV.9.3.289 16. Nakashima Y, Fujii H, Sumiyoshi S, Wight TN, Sueishi K. Early human atherosclerosis: accumulation of lipid and proteoglycans in intimal thickenings followed by macrophage infiltration. Arterioscler Thromb Vasc Biol 2007; 27:1159-65; PMID:17303781; http:// dx.doi.org/10.1161/ATVBAHA.106.134080 17. Little PJ, Ballinger ML, Osman N. Vascular wall proteoglycan synthesis and structure as a target for the prevention of atherosclerosis. Vasc Health Risk Manag 2007; 3:117-24; PMID:17583182 18. Cardoso LEM, Mour~ao PAS. Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb 1994; 14:115-24; PMID:8274466; http://dx.doi.org/10.1161/01. ATV.14.1.115 19. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits. II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis 1989; 9:908-18; PMID:2590068; http://dx.doi.org/10.1161/ 01.ATV.9.6.908 20. Davies JR, Rudd JH, Weissberg PL, Narula J. Radionuclide imaging for the detection of inflammation in vulnerable plaques. J Am Coll Cardiol 2006; 47 (Suppl):C57-68; PMID:16631511 21. Glaudemans AWJM, Slart RHJA, Bozzao A, Bonanno E, Arca M, Dierckx RAJO, Signore A. Molecular imaging in atherosclerosis. Eur J Nucl Med Mol Imaging 2010; 37:2381-97; PMID:20306036; http://dx.doi. org/10.1007/s00259-010-1406-4 22. Theocharis AD, Theocharis DA, De Luca G, Hjerpe A, Karamanos NK. Compositional and structural alterations of chondroitin and dermatan sulfates during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta. Biochimie 2002;
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23.
24.
25.
26.
27.
28.
29.
30.
84:667-74; PMID:12453639; http://dx.doi.org/ 10.1016/S0300-9084(02)01428-1 Theocharis AD, Tsolakis I, Tzanakakis GN, Karamanos NK. Chondroitin sulfate as a key molecule in the development of atherosclerosis and cancer progression. Adv Pharmacol 2006; 53:281-95; PMID:17239771; http://dx.doi.org/10.1016/S1054-3589(05)53013-8 Behr TM, Becker WS, Bair HJ, Klein MW, St€ uhler CM, Cidlinsky KP, Wittekind CW, Scheele JR, Wolf FG. Comparison of complete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for immunoscintigraphy in colorectal cancer. J Nucl Med 1995; 36:430-41; PMID:7884505 Sharkey RM, Karacay H, Cardillo TM, Chang CH, McBride WJ, Rossi EA, Horak ID, Goldenberg DM. Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods. Clin Cancer Res 2005; 11:7109s-21s; PMID:16203810; http://dx.doi. org/10.1158/1078-0432.CCR-1004-0009 Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, Ahmadi M, Thielens N, Wernery U, Caveliers V, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res 2012; 110:927-37; PMID:22461363; http://dx.doi.org/ 10.1161/CIRCRESAHA.112.265140 Hinojosa LE, Hernandez T, de Acosta CM, Montero E, Perez R, Lopez-Requena A. Construction of a recombinant non-mitogenic anti-human CD3 antibody. Hybridoma (Larchmt) 2010; 29:115-24; PMID:20443703; http:/ /dx.doi.org/10.1 089/ hyb.2009.0042 Mateo C, Moreno E, Amour K, Lombardero J, Harris W, Perez R. Humanization of a mouse monoclonal antibody that blocks the epidermal growth factor receptor: recovery of antagonistic activity. Immunotechnology 1997; 3:71-81; PMID:9154469; http://dx.doi.org/ 10.1016/S1380-2933(97)00065-1 Mather SJ, Ellison D. Reduction-Mediated Technetium-99m Labeling of Monoclonal Antibodies. J Nucl Med 1990; 31:692-7. Jellinek H, Harsing J, F€uzesi S. A new model for arteriosclerosis. An electron-microscopic study of the lesions induced by i.v. administered fat. Atherosclerosis 1982; 43:7-18; PMID:7092984; http://dx.doi.org/ 10.1016/0021-9150(82)90095-8
Volume 6 Issue 5
mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody a
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Yosdel Soto , Niurka Mesa , Yumisley Alfonso , Arlenis Pérez , Fernando Batlle , Tania a
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Griñán , Adonis Pino , Justo Viera , Milagros Frómeta , Victor Brito , Armando Olivera , b
Francisco Zayas & Ana M Vázquez a
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Research and Development Direction, Center of Molecular Immunology, Havana, Cuba
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Department of Nuclear Medicine, Institute of Nephrology, Havana, Cuba
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Parasitology Department, Institute of Tropical Medicine «Pedro Kourí», Havana, Cuba
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Experimental Surgery Department, Center of Medical and Surgical Research, Havana, Cuba Published online: 30 Oct 2014.
To cite this article: Yosdel Soto, Niurka Mesa, Yumisley Alfonso, Arlenis Pérez, Fernando Batlle, Tania Griñán, Adonis Pino, Justo Viera, Milagros Frómeta, Victor Brito, Armando Olivera, Francisco Zayas & Ana M Vázquez (2014) Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody, mAbs, 6:5, 1340-1346, DOI: 10.4161/mabs.29970 To link to this article: http://dx.doi.org/10.4161/mabs.29970
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REPORT mAbs 6:5, 1340--1346; September/October 2014; © 2014 Taylor & Francis Group, LLC
Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody ~an1, Adonis Pino3, Justo Viera1, Yosdel Soto1, Niurka Mesa2, Yumisley Alfonso2, Arlenis Perez1, Fernando Batlle2, Tania Grin 1 1 4 2 meta , Victor Brito , Armando Olivera , Francisco Zayas , and Ana M Vazquez1,* Milagros Fro 1
Research and Development Direction, Center of Molecular Immunology, Havana, Cuba; 2Department of Nuclear Medicine, Institute of Nephrology, Havana, Cuba; 3Parasitology Department, Institute of Tropical Medicine «Pedro Kourí», Havana, Cuba; 4Experimental Surgery Department, Center of Medical and Surgical Research, Havana, Cuba
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Keywords: monoclonal antibodies, glycosaminoglycans, atherosclerosis, technetium-99m, imaging Abbreviations: % ID/g, percentage of injected dose per gram of tissue; At-R, Atherosclerotic rabbits; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycans; DS, dermatan sulfate; ELISA, enzyme-linked immunoadsorbent assay; GAG, glycosaminoglycan; LDL, low density lipoprotein; mAb, monoclonal antibody; Non At-R, Non atherosclerotic rabbit; NZW rabbits, New Zealand White rabbits; PG, proteoglycans
The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n D 14) by the administration of Lipofundin 20% using PBSreceiving animals as control (n D 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of 99mTc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of 99mTc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, 99mTc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of 99mTc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.
Introduction Atherosclerosis is the underlying pathology of most cardiovascular events and the main cause of morbidity and mortality worldwide.1 This disease is characterized by a non-resolved inflammatory response triggered by the retention of apoB-containing lipoproteins in the artery wall.2,3 Upon entrapment in the intima layer, proatherogenic lipoproteins undergo oxidative and enzymatic modifications thereby increasing their immunogenicity.4 These modifications also trigger the uptake of lipoprotein by macrophages and vascular smooth muscle cells, which leads to foam cells formation.4,5 Subendothelial retention of low density lipoprotein (LDL) is mediated by electrostatic interactions between positively charged residues on apolipoprotein B-100 (apoB-100) and negatively
charged glycosaminoglycans (GAGs) chains on proteoglycans (PGs).6,7 A prominent feature of atherosclerosis progression is the accumulation of chondroitin sulfate (CS) and dermatan sulfate (DS) PGs, accompanied by hyperelongation of GAGs side chains.8,9 CSPG-LDL complexes have been detected in injured rabbit intima,10 and they have also been isolated from human atherosclerotic lesions.11 In a previous work, we characterized the antiatherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99, which binds sulfated GAGs, inhibits LDL binding to CS, and abrogates LDL oxidation in vitro. Moreover, rabbits immunized with chP3R99 mAb showed reduced aortic arch lesions and no macrophage infiltration following short-term treatment with Lipofundin 20% (referred to as Lipofundin).12 Similar results were observed in ApoE¡/¡ mice fed with a
*Correspondence to: Ana M Vazquez; Email: [email protected] Submitted: 04/01/2014; Revised: 07/09/2014; Accepted: 07/16/2014 http://dx.doi.org/10.4161/mabs.29970
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Figure 1. chP3R99 mAb reactivity to Lipofundin-induced atherosclerotic plaque from rabbit aorta. GAGs were detected in the artery wall of normal and Lipofundin-treated rabbits by Alcian Blue staining and immunohistochemistry with CS-56 mAb (1 mg/mL). Samples from normal aorta and sections corresponding to areas with early and advanced lesions were incubated with chP3R99 mAb or the isotype-matched control hR3 (2 mg/mL). Original magnification X20. Scale bars D 100 mm.
high-fat high-cholesterol diet in which the administration of chP3R99 mAb reduced the total lesion area in more than 40%.13 In this paper, we demonstrated that chP3R99 mAb preferentially accumulates in rabbit atherosclerotic lesions, supporting the use of anti-GAGs antibodies for the detection and treatment of atherosclerosis.
Results chP3R99 mAb reactivity to rabbit atherosclerotic lesions To evaluate the content of GAG in the artery wall, aortic sections from normal and Lipofundin-treated rabbits were stained with Alcian Blue. As depicted in Figure 1, the amounts of GAGs increased in the aortic arch depending on the presence and size of the lesions. Particularly, immunohistochemistry revealed a high content of CS in the aortic lesions of Lipofundin-receiving rabbits, which results in a strong reactivity of chP3R99 mAb in these areas. A lower reactivity was observed in aortic sections from the control rabbits and in Type I AHA lesions, whereas the isotypematched control (hR3 mAb) did not react with any of the aortic sections.
intensity of the fluorescence were lower in the aorta of healthy animals, and located predominantly in the adventitia (Fig. 2D–F). In fact, as depicted in Figure 2J, the accumulation of chP3R99 was significantly higher (8.7-fold) in atherosclerotic rabbits than in controls; and also higher (135-fold) than the accumulation of the isotype-matched mAb in atherosclerotic rabbits (P < 0.05). These results were confirmed by immunoscintigraphy by intravenous administration of 99mTc-chP3R99 mAb in atherosclerotic and control rabbits (Fig. 3). Ten minutes after the injection of 99mTc-chP3R99 mAb or the isotype-matched control 99m Tc-chT3 mAb, blood-pool images were similar in all animals (data not shown). The overall uptake was predominantly localized in liver, kidneys and heart alongside all time intervals. Planar images acquisition revealed the accumulation of 99mTc-chP3R99 mAb in the carotid of atherosclerotic rabbits 6 h after radiotracer administration (Fig. 3A), but not in control animals (Fig. 3B). The visualization of atherosclerotic lesions upon 99mTc-chP3R99 mAb injection was specific, since no evident accumulation of 99m Tc-chT3 mAb was observed in Lipofundin-receiving rabbits (Fig. 3C). 99m
chP3R99 mAb accumulation in Lipofundin-induced lesions The accumulation of chP3R99 mAb in atherosclerotic lesions in vivo was addressed by immunofluorescence upon intravenous injection of 1 mg of the mAb in rabbits receiving or not Lipofundin (Fig. 2). Unlike hR3 mAb (Fig. 2A–C), chP3R99 was detected in lesioned arterial tissue, adventitia and the surface of smooth muscle cells of atherosclerotic rabbits (Fig. 2G–I). In contrast, both the presence of this anti-GAGs mAb and the
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Tc-chP3R99 mAb arterial uptake The distribution of 99mTc-chP3R99 mAb in rabbits is summarized in Figure 4. The percentage of injected dose of the radiotracer per gram of tissue (% ID/g) in samples of Lipofundin-treated rabbits was greater in kidney (21.3 § 1.8% ID/g) and urine (15.7 § 6.0% ID/g). As depicted in Figure 4A and 4B, we found similar mAb uptake by non-targeted organs without marked differentiation between lesioned and non-lesioned rabbits, (P > 0.05). In contrast, 99m Tc-chP3R99 mAb accumulation into atherosclerotic lesions was
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greater than the one observed in the artery wall of control rabbits, both for aortic arch (1.019 § 0.294% ID/g vs. 0.187 § 0.097% ID/g) and thoracic segment (0.547 § 0.180% ID/g vs. 0.097 § 0.035% ID/g), (P < 0.05). In these segments, the accumulation of radiolabeled mAb was more than 5-fold higher in Lipofundin-receiving rabbits than in controls (Fig. 4B). According to immunoscintigraphy images, we 99m Figure 2. Immunofluorescence detection of in vivo chP3R99 accumulation in Lipofundin-induced atherosclerotic found that Tclesions. Representative images from atherosclerotic rabbits injected with 1 mg of the isotype matched control (A–C) chP3R99 mAb uptake by or chP3R99 mAb (G–I). Frozen sections from healthy rabbits who received the same mass of chP3R99 are shown (D–F). carotids with lesions was Human antibodies were detected through the incubation with a goat phycoerythrine (PE)-conjugated anti-human IgG 3.9-fold higher than that followed by counterstaining with Hoechst. Original magnification X20. Scale bars D 100 mm. (J) Represents the percentages of human IgG stained areas respect to total area. At-R D atherosclerotic rabbits; NAt-R D non atherosclerotic rabin controls (0.597 § bits. Data are expressed as mean § SD. 0.079% ID/g vs. 0.157 § 0.140% ID/g), (P < 0.05). Although the % ID/g in abdominal portion of aorta from Discussion Lipofundin-receiving animals was 2.8-fold higher than in non-athThe use of therapeutic agents targeting the arterial wall, espeerosclerotic rabbits, no significant differences were observed between these groups (0.356 § 0.174 ID/g vs. 0.139 § 0.121% cially those aimed at preventing the retention of proatherogenic ID/g), (P > 0.05). Also, chP3R99 mAb accumulation in aortic arch from rabbits with atheromatous plaques was 1.7-fold and 2.5-fold higher than in thoracic and abdominal segments, respectively. In addition, the % ID/g ratio of arterial segments-to-muscle and arterial segments-toblood were significantly greater in Lipofundin-receiving rabbits than in controls (P < 0.05) (Table 1). Histopathological study 99m Tc-chP3R99 mAb accumulation in different segments of the aorta and carotids was associated to lesions size of Lipofundinreceiving rabbits (Fig. 5). Histopathological studies revealed the presence of atherosclerotic lesions in aortic arch (Fig. 5A and 5B) and thoracic segment (Fig. 5C) of aorta and carotids (Fig. 5D), characterized by intimal thickening, leukocyte infiltration and distortion of smooth muscle cells arrangement. In contrast, only a slight thickening of the intima was observed in the abdominal segment (Fig. 5E). The aortas from PBS-receiving rabbits showed no alteration in the vascular architecture (Fig. 5F).
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Figure 3. Immunoscintigrams of rabbits injected with 99mTc-chP3R99 mAb or 99mTc-chT3 mAb. Planar images acquired at 6 h after radiotracers injection, showed a selective accumulation of chP3R99 mAb in carotids (arrowhead) from rabbits with Lipofundin-induced atherosclerotic lesions (A) but not in healthy rabbits (B). No accumulation of the isotype-matched control mAb was observed in atherosclerotic lesions (C).
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reduce lesions size or to halt their growth. In rabbits, the acute administration of Lipofundin induces American Heart Association type IIIIV atherosclerotic lesions characterized by accumulation of abundant GAGs and collagen fibers in the subendothelial space, along with macrophages infiltration.12 According to the in vitro results, immunoscintigraphy and immunofluorescence analysis revealed a preferential accumulation of chP3R99 mAb in atherosclerotic plaques in rabbits. In Figure 4. Biodistribution of 99mTc-chP3R99 mAb in rabbits determined as % ID/g 6 h after radiotracer administracontrast to the isotypetion. (A) Radiotracer uptake by organs. Black and white bars represent the % ID/g in atherosclerotic and healthy matched control, chP3R99 rabbits, respectively. (B) Ratio of 99mTc-chP3R99 mAb uptake by samples from rabbits with Lipofundin-induced athmAb showed sustained accuerosclerosis to non-atherosclerotic rabbits. Data are expressed as mean § SD. At-R D atherosclerotic rabbits; NAt-R mulation within carotids D non atherosclerotic rabbits. from Lipofundin-treated rabbits, but not in the arterlipoproteins in the subendothelial matrix, is a promising strategy ies of control animals, resulting in a specific uptake by atherosclewhich has been poorly addressed.14 Previously, we reported that rotic lesions. These results agreed with a higher detection of the chP3R99 mAb prevents atherosclerosis in rabbits; presumably chimeric antibody by immunofluorescence in frozen aortic secthrough the induction of autologous anti-CS antibodies gener- tions from rabbits with lesions. In the same line, Brito and cowated upon immunization. However, a direct proof of the reactiv- orkers reported that chP3R99-fluorescein isothiocyanate injected ity of chP3R99 mAb or those antibodies with the rabbit artery intravenously colocalizes with activated endothelial areas of aorta wall was not demonstrated.12 Now, we showed that chP3R99 in ApoE¡/¡ mice fed a high-fat high-cholesterol diet.13 mAb specifically recognizes atherosclerotic lesions, in a close assoDue to the small size of atherosclerotic plaques and the close ciation with their size and the presence of CS GAGs. The devel- proximity of aorta to heart and liver we could not visualize this opment of atherosclerosis is characterized by a shifting of artery by immunoscintigraphy. Notwithstanding, as revealed in contractile vascular smooth muscle cells toward secretory pheno- biodistribution studies, the uptake of 99mTc-chP3R99 mAb by type, thereby affecting the composition and atherogenicity of aortic arch and thoracic segment from atherosclerotic rabbits was PGs produced.15 For instance, it have been described a promi- almost 6-folds higher than by the arterial wall of healthy animals. nent accumulation of CSPGs, throughout the artery wall16 with In addition, radiotracer uptake was not evenly distributed elongated GAGs chains and higher sulfation degree at atheroscle- throughout the descending aorta and was related with the size of lesions detected in these areas. Indeed, 99mTc-chP3R99 accumurosis-prone sites.17,18 Hence, these results might suggest the feasibility of using this lation in the aortic cross was 2.5-fold greater than in abdominal anti-GAG mAb in advanced stages of the disease in order to segment in atherosclerotic rabbits. Although it has been described that LDL retention in the artery wall is similar among all aortic sites of normal rabbits, it is increased at atherosclerosis-susceptible areas as soon as 4 d after feeding with hypercholesterolemic Table 1. Biodistribution of chP3R99 mAb in arterial segments diet.19 Control rabbits Atherosclerotic rabbits Here, we also demonstrated that Lipofundin, in addition to (n D 3) (n D 4) develop atherosclerosis in aorta, also induces lesions in carotids as Arterial Segment A/M A/B A/M A/B reflected by the thickening of the intima layer, extracellular matrix production and distortion in the arrangement of smooth Aortic Arch 0.47 § 0.26 0.14 § 0.12 7.09 § 1.12* 0.93 § 0.36* 99m TcThoracic 0.80 § 0.30 0.09 § 0.07 5.12 § 1.59* 0.50 § 0.32* muscle cells. Thus, we found almost a 4-folds increase in chP3R99 mAb uptake into this artery, supporting the accumulaAbdominal 0.55 § 0.09 0.16 § 0.12 2.34 § 0.08* 0.55 § 0.38 Carotids 0.77 § 0.19 0.27 § 0.18 2.67 § 0.35* 0.75 § 0.19* tion of the mAb observed by immunoscintigraphy. Until now, most of the efforts in the development of radioValues are expressed as mean § SD. A/M: arteria-to-muscle ratio; tracers to detect atherosclerotic lesions have been focused on the A/B: arteria-to-blood ratio; *P < 0.05 vs. control group. identification of important pathways associated with plaque
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instability, such as macrophage recruitment, matrix breakdown enzyme, macrophage metabolism, thrombosis and apoptosis.20,21 However, since CS/DS PGs are produced in large quantities during all the stages of human atherosclerosis development they could be a good target to detect atherosclerotic lesions, even in early phases of the disease.22,23 This could be useful in atherosclerosis diagnosing as a criterion to prescribe artery wall-based therapies.17 Because we used as radiotracer a full-sized mAb tagged with 99Tc, there was a slow blood clearance of the probe, resulting in a suboptimal target-to-background ratio. Hence, the 99m Tc-chP3R99 mAb uptake detected by immunoscintigraphy in the lesioned carotids could be attributable to both specific accumulation and blood pool radiactivity. However, it was possible to observe a focal accumulation of chP3R99 mAb in some regions of carotids from Lipofundin-receiving rabbits along with higher A/B and A/M ratios in these animals. Conversely, we could not detect neither chP3R99 mAb in carotids from healthy animals nor accumulation of the isotype-matched control mAb in atherosclerotic plaques. Future studies using fragments of chP3R99 mAb (Fab, scFv) or engineered variants (diabodies, triabodies, minibodies, and single-domain antibodies) are required Figure 5. Representative atherosclerotic lesions developed in aortic segments and carotids of in order to optimize the targeting of atheroLipofundin-receiving rabbits. Hematoxylin-Eosin shows intimal thickening, leukocyte infiltration sclerotic lesions with a fast clearance from and distortion in the vascular tissue architecture in aortic arch (A, B), thoracic segment (C) and circulation.24,25 A recent approach for carcarotids (D) of Lipofundin-treated rabbits. Minor lesions were observed in the abdominal segment of aorta (E) whereas no alteration in the artery wall of normal rabbits was detected (F). diovascular applications is the use of nanoOriginal magnification X20 except for B (X40). Scale bars D 50 mm except for (D) where bodies as radiotracers, displaying high bar D 100 mm. stability, fast blood clearance, and the possibility of labeling with different probes thereby allowing atherosclerotic lesions imaging.26 of Laboratory Animals (CENPALAB, Mayabeque, Cuba). AniIn summary, our results show that chP3R99 mAb preferen- mals were housed under standard conditions (25 C, 60 § 10% tially accumulates in atherosclerotic lesions, even at early phases humidity), 12 h day/night cycles with water and food ad libitum. of the disease, supporting the potential use of this anti-GAGs All procedures were approved by the Institutional Animal Ethic antibody for diagnosing and treatment of atherosclerosis. Committee.
Materials and Methods Antibodies chP3R99, chT3 (anti-human CD3)27 and hR3 (anti-Epidermal Growth Factor receptor)28 mAbs were purified by protein-A affinity chromatography (Pharmacia) from transfectoma culture supernatants and analyzed by SDS-PAGE. Animals New Zealand White (NZW) male rabbits (3.0–3.5 kg, 12 wk old) were obtained from the National Center for the Production
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Radiolabeling of mAbs Labeling of mAbs with 99mTc was performed according to a method previously reported.29 Briefly, mAbs were reduced at a 2-mercaptoethanol-to-mAb molar ratio of 850:1 at room temperature (RT) for 30 min. After purification by a PD-10 column chromatography (Sephadex G-25), the reactivity of the reduced mAbs to CS (Sigma) was determined by ELISA, as previously described.12 Then, 84 mg of sodium pyrophosphate and 2 mg of stannous chloride were diluted in 5.0 mL of sodium chloride 0.9% and 100 mL of this mixture were added to 1 mg of the reduced mAbs. The samples were incubated for 15 min at RT followed by a 30 min incubation with 1295 MBq (35 mCi)
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99m
Tc pertechnetate eluted from a 99mMo–99mTc generator (CENTIS, Mayabeque, Cuba). Quality control was determined by thin-layer chromatography using both acetone and sodium chloride 0.9% as mobile phase yielding >90% of radiochemical purity.
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Cysteine and rabbit sera challenge Transchelation experiments were performed by challenging 31 mg of 99mTc-chP3R99 mAb with 630 nmol of cysteine at a final molar ratio cysteine to mAb 3000:1, and rabbit sera (volume ratio 20:1) at RT for 24 h. In these extreme conditions we found 40–50% of displacement of the radioactivity. Accumulation of chP3R99 mAb in atherosclerotic lesions Atherosclerosis was induced in 18 NZW rabbits by daily administration of Lipofundin 20% (Braun Melsungen) (2 mL/ kg, 8 d) through marginal ear vein, as previously described.12,30 Eight rabbits that received only PBS were used as control. To evaluate the preferential accumulation of chP3R99 mAb in atherosclerotic lesions, six Lipofundin-treated rabbits and four healthy controls intravenously received 1mg of this antibody into the marginal ear vein the day after the last dose of Lipofundin and were sacrificed 24 h later. Specificity was assessed by administrating the same mass of hR3 mAb in four atherosclerotic rabbits. In other set of experiments, 99mTc-chP3R99 mAb (0.3 mg, 370 MBq) was injected into the marginal ear vein both in Lipofundin-treated (n D 4) and PBS-receiving rabbits (n D 3). As a control of specificity, two atherosclerotic rabbits received the same dose of 99mTc-chT3 mAb. After 12 h of fasting, rabbits were anesthetized with 35 mg/kg of ketamine hydrochloride and 5 mg/kg of xylazine HCl, IM. Radioimmunoscintigraphy was performed 10 min after radiotracers injection and repeated 6 h later. Planar images were obtained in the anterior views with a gamma camera (MEDISO) equipped with a low-energy, parallelhole, high-resolution collimator and stored on a 128 £ 128 pixel matrix by means of a digital computer. Images were recorded until 200K counts at the energy peak of 140 keV with a 20% window. Rabbits were sacrificed by intracoronary injection of KCl under anesthesia and the following organs and tissues were removed, weighed, and counted in a well scintillation g-counter (CAPINTEC, Inc. CRC-25W): aorta (aortic arch, thoracic and abdominal segments), carotids, heart, lung, liver, spleen, kidney and muscle. The radioactivity of blood and urine was counted as well. Radioactivity uptake was expressed as percentage of injected dose per gram of tissue (% ID/g). Specific antibody accumulation in organs was considered when the ratio of % ID/g of atherosclerotic-to-control rabbits was >2.5. References 1. Negi S, Anand A. Atherosclerotic coronary heart disease-epidemiology, classification and management. Cardiovasc Hematol Disord Drug Targets 2010; 10:257-61; PMID:20932265; http://dx.doi.org/ 10.2174/187152910793743832 2. Tabas I, Glass CK. Anti-inflammatory therapy in chronic disease: challenges and opportunities. Science 2013;
www.landesbioscience.com
Histological study Histopathological findings in the different segments of aorta and carotids as well as immunohistochemical studies were performed in formalin-fixed (24 h, pH 7.4) paraffin-embedded tissue samples. The specimens were cut into 5-mm slices, deparaffinized, rehydrated, and stained with hematoxylin-eosin or Alcian Blue for light microscopic examination. The reactivity to the artery wall was evaluated in aortic sections from two rabbits in which the atherosclerotic lesions were induced or unmanipulated ones. After blocking endogenous peroxidase activity (Dako), sections were incubated with chP3R99 mAb or the isotype-matched control hR3 (2 mg/mL) for 1 h at RT, followed by an anti-human IgG serum conjugated to peroxidase (Jackson ImmunoResearch Lab). Anti-CS mAb (clone CS-56, Sigma) was used as positive control (1 mg/mL) and it was identified by the incubation with the EnVisionTM Detection Systems (Dako). Finally, sections were incubated with diaminobenzidine substrate solution and counterstained with hematoxylin. The presence of chP3R99 mAb in the artery wall was detected by immunofluorescence in fresh frozen samples, by incubating tissue sections with a goat phycoerythrine (PE)-conjugated antihuman IgG (BD Bioscience), followed by counterstaining with Hoechst. Images were digitally captured with a DP20 camera coupled to a fluorescence microscope Olympus BX51. Fluorescence images were quantified by using the software ImageJ 1.47. Statistical analysis Data are expressed as the mean § SD. To compare the accumulation of the chimeric antibodies in the artery wall of rabbits in immunofluorescence experiments a one-way ANOVA was performed followed by Bonferroni post hoc test. The % ID/g in organs and ratios between atherosclerotic rabbits and controls were compared by use of the Student t test. A P < 0.05 value was considered statistically significant.
Disclosure of Potential Conflicts of Interest
Ana Marıa Vazquez, Yosdel Soto and Victor Brito are inventors of a patent related with antibodies that recognize sulfatides and sulfated PGs; however, they have assigned their rights to the assignee Center of Molecular Immunology.
Acknowledgments
This work was supported by the Center of Molecular Immunology (Havana, Cuba).
339:166-72; PMID:23307734; http://dx.doi.org/ 10.1126/science.1230720 3. Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 2013; 339:161-6; PMID:23307733; http://dx. doi.org/10.1126/science.1230719 4. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol
mAbs
2006; 6:508-19; PMID:16778830; http://dx.doi.org/ 10.1038/nri1882 5. Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation 2007; 116:1832-44; PMID:17938300; http://dx.doi.org/ 10.1161/CIRCULATIONAHA.106.676890 6. Hurt-Camejo E, Olsson U, Wiklund O, Bondjers G, Camejo G. Cellular consequences of the association of
1345
7.
8.
Downloaded by [University of Western Ontario] at 02:33 05 February 2015
9.
10.
11.
12.
13.
apoB lipoproteins with proteoglycans. Potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol 1997; 17:1011-7; PMID:9194748; http://dx.doi.org/ 10.1161/01.ATV.17.6.1011 Khalil MF, Wagner WD, Goldberg IJ. Molecular interactions leading to lipoprotein retention and the initiation of atherosclerosis. Arterioscler Thromb Vasc Biol 2004; 24:2211-8; PMID:15472124; http://dx.doi.org/ 10.1161/01.ATV.0000147163.54024.70 Little PJ, Osman N, O’Brien KD. Hyperelongated biglycan: the surreptitious initiator of atherosclerosis. Curr Opin Lipidol 2008; 19:448-54; PMID:18769225; http:// dx.doi.org/10.1097/MOL.0b013e32830dd7c4 Ballinger ML, Osman N, Hashimura K, de Haan JB, Jandeleit-Dahm K, Allen T, Tannock LR, Rutledge JC, Little PJ. Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo. J Cell Mol Med 2010; 14(6B):1408-18; PMID:19754668; http://dx. doi.org/10.1111/j.1582-4934.2009.00902.x Srinivasan SR, Vijayagopal P, Dalferes ERJ Jr., Abbate B, Radhakrishnamurthy B, Berenson GS. Dynamics of lipoprotein-glycosaminoglycan interactions in the atherosclerotic rabbit aorta in vivo. Biochim Biophys Acta 1984; 793:157-68; PMID:6712964; http://dx.doi.org/ 10.1016/0005-2760(84)90317-5 Camejo G, Hurt E, Romano M. Properties of lipoprotein complexes isolated by affinity chromatography from human aorta. Biomed Biochim Acta 1985; 44:389-401; PMID:4004839 Soto Y, Acosta E, Delgado L, Perez A, Falcon V, Becquer Brito V, Alvarez MA, Fraga A, I, Gri~ nan T, et al. Antiatherosclerotic effect of an antibody that binds to extracellular matrix glycosaminoglycans. Arterioscler Thromb Vasc Biol 2012; 32:595-604; PMID:22267481; http://dx.doi. org/10.1161/ATVBAHA.111.238659 Brito V, Mellal K, Giroux S, Perez A, Soto Y, deBlois D, Ong H, Marleau S, Vazquez AM. Induction of anti-antiidiotype antibodies against sulfated glycosaminoglycans reduces atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2012; 32:2847-54; PM ID:2 3087361; http:/ /dx.doi.org/10 .1161/ ATVBAHA.112.300444
1346
14. Tabas I. Making things stick in the fight against atherosclerosis. Circ Res 2013; 112:1094-6; PMID:23580771; http://dx.doi.org/10.1161/CIRCRESAHA.113.301227 15. Manderson JA, Mosse PRL, Safstrom JA, Young SB, Campbell GR. Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype. Arteriosclerosis 1989; 9:289-98; PMID:2719591; http:// dx.doi.org/10.1161/01.ATV.9.3.289 16. Nakashima Y, Fujii H, Sumiyoshi S, Wight TN, Sueishi K. Early human atherosclerosis: accumulation of lipid and proteoglycans in intimal thickenings followed by macrophage infiltration. Arterioscler Thromb Vasc Biol 2007; 27:1159-65; PMID:17303781; http:// dx.doi.org/10.1161/ATVBAHA.106.134080 17. Little PJ, Ballinger ML, Osman N. Vascular wall proteoglycan synthesis and structure as a target for the prevention of atherosclerosis. Vasc Health Risk Manag 2007; 3:117-24; PMID:17583182 18. Cardoso LEM, Mour~ao PAS. Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb 1994; 14:115-24; PMID:8274466; http://dx.doi.org/10.1161/01. ATV.14.1.115 19. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits. II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis 1989; 9:908-18; PMID:2590068; http://dx.doi.org/10.1161/ 01.ATV.9.6.908 20. Davies JR, Rudd JH, Weissberg PL, Narula J. Radionuclide imaging for the detection of inflammation in vulnerable plaques. J Am Coll Cardiol 2006; 47 (Suppl):C57-68; PMID:16631511 21. Glaudemans AWJM, Slart RHJA, Bozzao A, Bonanno E, Arca M, Dierckx RAJO, Signore A. Molecular imaging in atherosclerosis. Eur J Nucl Med Mol Imaging 2010; 37:2381-97; PMID:20306036; http://dx.doi. org/10.1007/s00259-010-1406-4 22. Theocharis AD, Theocharis DA, De Luca G, Hjerpe A, Karamanos NK. Compositional and structural alterations of chondroitin and dermatan sulfates during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta. Biochimie 2002;
mAbs
23.
24.
25.
26.
27.
28.
29.
30.
84:667-74; PMID:12453639; http://dx.doi.org/ 10.1016/S0300-9084(02)01428-1 Theocharis AD, Tsolakis I, Tzanakakis GN, Karamanos NK. Chondroitin sulfate as a key molecule in the development of atherosclerosis and cancer progression. Adv Pharmacol 2006; 53:281-95; PMID:17239771; http://dx.doi.org/10.1016/S1054-3589(05)53013-8 Behr TM, Becker WS, Bair HJ, Klein MW, St€ uhler CM, Cidlinsky KP, Wittekind CW, Scheele JR, Wolf FG. Comparison of complete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for immunoscintigraphy in colorectal cancer. J Nucl Med 1995; 36:430-41; PMID:7884505 Sharkey RM, Karacay H, Cardillo TM, Chang CH, McBride WJ, Rossi EA, Horak ID, Goldenberg DM. Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods. Clin Cancer Res 2005; 11:7109s-21s; PMID:16203810; http://dx.doi. org/10.1158/1078-0432.CCR-1004-0009 Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, Ahmadi M, Thielens N, Wernery U, Caveliers V, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res 2012; 110:927-37; PMID:22461363; http://dx.doi.org/ 10.1161/CIRCRESAHA.112.265140 Hinojosa LE, Hernandez T, de Acosta CM, Montero E, Perez R, Lopez-Requena A. Construction of a recombinant non-mitogenic anti-human CD3 antibody. Hybridoma (Larchmt) 2010; 29:115-24; PMID:20443703; http:/ /dx.doi.org/10.1 089/ hyb.2009.0042 Mateo C, Moreno E, Amour K, Lombardero J, Harris W, Perez R. Humanization of a mouse monoclonal antibody that blocks the epidermal growth factor receptor: recovery of antagonistic activity. Immunotechnology 1997; 3:71-81; PMID:9154469; http://dx.doi.org/ 10.1016/S1380-2933(97)00065-1 Mather SJ, Ellison D. Reduction-Mediated Technetium-99m Labeling of Monoclonal Antibodies. J Nucl Med 1990; 31:692-7. Jellinek H, Harsing J, F€uzesi S. A new model for arteriosclerosis. An electron-microscopic study of the lesions induced by i.v. administered fat. Atherosclerosis 1982; 43:7-18; PMID:7092984; http://dx.doi.org/ 10.1016/0021-9150(82)90095-8
Volume 6 Issue 5
