Antiangiogenic Synergism of Integrin-Targeted Fumagillin Nanoparticles and Atorvastatin in Atherosclerosis
Patrick M. Winter, PhD*,
Shelton D. Caruthers, PhD*, ,
Huiying Zhang, MD*,
Todd A. Williams, RT, MR*,
Samuel A. Wickline, MD, FACC*,
Gregory M. Lanza, MD, PhD, FACC*,*
* Washington University, St. Louis, Missouri
Philips Healthcare, Andover, Massachusetts
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Abstract
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Objectives: Studies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic   β3-targeted nanoparticles.
Background: Antiangiogenesis therapy may normalize atherosclerotic plaque vasculature and promote plaque stabilization. β3-targeted paramagnetic nanoparticles can quantify atherosclerotic angiogenesis and incorporate fumagillin to elicit acute antiangiogenic effects.
Methods: In the first experiment, hyperlipidemic rabbits received β3-targeted fumagillin nanoparticles (0, 30, or 90 µg/kg) with either a continued high fat diet or conversion to standard chow. The antiangiogenic response was followed for 4 weeks by cardiac magnetic resonance (CMR) molecular imaging with β3-targeted paramagnetic nanoparticles. In a second 8-week study, atherosclerotic rabbits received atorvastatin (0 or 44 mg/kg diet) alone or with β3-targeted fumagillin nanoparticles (only week 0 vs. weeks 0 and 4), and angiogenesis was monitored with CMR molecular imaging. Histology was performed to determine the location of bound nanoparticles and to correlate the level of CMR enhancement with the density of angiogenic vessels.
Results: The β3-targeted fumagillin nanoparticles reduced the neovascular signal by 50% to 75% at 1 week and maintained this effect for 3 weeks regardless of diet and drug dose. In the second study, atherosclerotic rabbits receiving statin alone had no antineovascular benefit over 8 weeks. The β3-targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks as in study 1, and readministration on week 4 reproduced the 3-week antineovascular response with no carry-over benefit. However, atorvastatin and 2 doses of β3-targeted fumagillin nanoparticles (0 and 4 weeks) achieved marked and sustainable antiangiogenesis. Microscopic studies corroborated the high correlation between CMR signal and neovessel counts and confirmed that the β3-targeted nanoparticles were constrained to the vasculature of the aortic adventia.
Conclusions: The CMR molecular imaging with β3-targeted paramagnetic nanoparticles demonstrated that the acute antiangiogenic effects of β3-targeted fumagillin nanoparticles could be prolonged when combined with atorvastatin, representing a potential strategy to evaluate antiangiogenic treatment and plaque stability.
Key Words: angiogenesis • molecular imaging • fumagillin • nanoparticle
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Abbreviations and Acronyms
| | AlkP = alkaline phosphatase | | ALT = alanine aminotransferase | | AST = aspartate aminotransferase | | CMR = cardiac magnetic resonance | | GGT = gamma glutamyltransferase | | MetAP = methionine aminopeptidase |
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Atherosclerotic plaque progresses from an early atheromatous lesion to a thin-capped vulnerable plaque through aggressive inflammatory and immune responses, comprising macrophage infiltration with necrotic core enlargement, neovascular expansion of the vasa vasorum, and intraplaque hemorrhage (1–3). Increased plaque angiogenesis, driven by hypoxia (4), proangiogenic growth factors (5), and oxidative stress (6), portends unstable vascular disease (1,2). Angiogenesis is correlated with plaque rupture (1) and is associated with the morphological features of vulnerable atheromas, including macrophage-infiltrated fibrous caps (1), lipid-rich cores (7), and thin-cap shoulders (1). The preponderance of data from experimental models and human pathological samples indicates that plaque neovasculature could serve as a molecular imaging biomarker of atherosclerotic severity and cardiovascular disease risk.
Traditional therapies, such as 3-hydroxy-3-methyl-glutaryl–coenzyme A reductase inhibitors, i.e., statins, have been proven to reduce cardiovascular risk (8), but their benefits may exceed the primary lipid-lowering effects. The pleiotropic benefits of statins have been attributed to antioxidant effects (9), diminished leukocyte–endothelial cell adhesion (10), attenuated macrophage activation and cytokine release (11), increased endothelial nitric oxide activity (12), or atherosclerotic plaque stabilization (13). Pathological data from excised carotid arteries of patients treated for 3 months with statins have revealed a reduction in microvascular density, which was proposed as an explanation for the additional benefit of statins (14). Others have asserted that direct evidence of decreased intraplaque angiogenesis attributable to statins and clinical improvement is lacking, and a normalized vasculature achieved through neovascular pruning may be less prone to intraplaque hemorrhage and may promote plaque stabilization (3,15).
Three antivascular endothelial growth factor drugs have been approved for use in specific cancers, but these drugs are expensive and have significant adverse effects, including proteinuria, hypertension, thrombosis, and intestinal perforation (16). Although this risk-benefit profile is acceptable in the context of cancer patient survival, the efficacy of these or similar drugs for the chronic treatment of patients with atherosclerosis is less clear. A potent clinically translatable antiangiogenic strategy that provides direct monitoring and treatment is needed for the chronic management of atherosclerotic patients.
We have shown that β3-targeted paramagnetic nanoparticles can be used to quantify angiogenesis in atherosclerosis (17), and with the incorporation of fumagillin this theranostic (i.e., therapeutic and diagnostic) agent can deliver an acute antiangiogenic effect (18). The overarching hypothesis of these studies was to determine whether the acute benefits of β3-targeted fumagillin nanoparticles could be incorporated into a clinically translatable regimen for testing the potential benefits of long-term antiangiogenesis therapy. Accordingly, the first objective of these studies was to define the antiangiogenic pharmacodynamics of a single β3-targeted fumagillin nanoparticle dosage. The second objective was then to determine if the antiangiogenic effects of β3-targeted fumagillin nanoparticles used acutely could be sustained with oral atorvastatin.
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Methods
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β3-targeted nanoparticle synthesis.
The β3-targeted nanoparticles were prepared similar to previous reports (17–19). All nanoparticle emulsions comprised 20% (v/v) perfluorooctylbromide (Exfluor, Round Rock, Texas), 1.3% to 2% (w/v) of a surfactant comixture, 1.7% (w/v) glycerin, and water for the balance. The surfactant comixture for the paramagnetic nanoparticles included 69.9 mol% lecithin (Avanti Polar Lipids, Alabaster, Alabama), 0.1 mol% peptidomimetic vβ3-integrin antagonist (U.S. patent 6,322,770) conjugated to polyethylene glycol2,000-phosphatidylethanolamine (Avanti Polar Lipids) and 30 mol% gadolinium diethylene-triamine-pentaacetic acid–bis-oleate (Gateway Chemical Technologies, St. Louis, Missouri). The fumagillin (Sigma, St. Louis, Missouri) was included at the proportionate expense of lecithin in the surfactant at 2 different levels corresponding to 30 or 90 µg of drug per milliliter of emulsion. The β3-targeted rhodamine nanoparticles incorporated 0.1 mol% rhodamine phosphatidylethanolamine in the surfactant mix at the expense of lecithin for fluorescent microscopy studies.
The vβ3-integrin antagonist used is a quinalone nonpeptide developed by Bristol-Myers Squibb Medical Imaging (U.S. patent 6,511,648), initially reported as the 111In–1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetic acid conjugate RP748 and cyan 5.5 homologue TA145 (20). The vβ3-ligand has a 15-fold preference for the Mn2+-activated receptor (21 nmol/l) (20) and a one-half maximal inhibitory concentration (IC50) for vβ5, 5β1, and glycoprotein IIb/IIIa of >10 µmol/l (Bristol-Myers Squibb Medical Imaging, Billerica, Massachusetts; DS Edwards and TD Harris, unpublished data, November, 2002). Nanoparticles have an IC50 of 50 pmol/l for the Mn2+-activated vβ3-integrin (Kereos, St. Louis, Missouri; JL Keene and RA Beardsley, unpublished data, May, 2007).
Experimental design.
All animal care and experimental protocols were in accordance with Washington University guidelines. New Zealand White rabbits (n = 37; 10 to 12 weeks old; Charles River Laboratories, Wilmington, Massachusetts) were fed a 0.25% cholesterol diet (Purina Mills, St. Louis, Missouri) for 100 days. In 1 study, animals either continued on the high-cholesterol diet or were converted to standard chow after the targeted fumagillin treatment; however, the serum cholesterol levels remained highly elevated over the course of the study in all of the animals (1,374 ± 67 mg/dl) independent of treatment. Therefore, in the second study, the high-cholesterol diet was continued with or without atorvastatin alone or in combination with nanoparticle treatment.
Study 1
The hypothesis tested in study 1 was whether a single pulsed dose of β3-targeted fumagillin nanoparticles could persistently suppress angiogenesis in the aortic wall. Animals were randomly assigned to 1 of 6 treatment groups: - 1 β3-targeted paramagnetic nanoparticles without fumagillin and 0.0% cholesterol (n = 6).
- 2 β3-targeted paramagnetic nanoparticles without fumagillin and 0.25% cholesterol (n = 8).
- 3 β3-targeted paramagnetic nanoparticles with fumagillin (30 µg/kg body weight) and 0.0% cholesterol (n = 7).
- 4 β3-targeted paramagnetic nanoparticles with fumagillin (30 µg/kg body weight) and 0.25% cholesterol (n = 6).
- 5 β3-targeted paramagnetic nanoparticles with fumagillin (90 µg/kg body weight) and 0.0% cholesterol (n = 5).
- 6 β3-targeted paramagnetic nanoparticles with fumagillin (90 µg/kg body weight) and 0.25% cholesterol (n = 5).
Therapeutic or control nanoparticles were administered IV on week 0 and cardiac magnetic resonance (CMR) molecular imaging of angiogenesis was repeated on weeks 1, 2, 3, and 4 using β3-targeted paramagnetic nanoparticles (no drug).
Study 2
The hypothesis tested in study 2 was whether combinational therapy with β3-targeted fumagillin nanoparticles and atorvastatin could sustain the antiangiogenic effect observed in study 1. Rabbits were maintained on the 0.25% cholesterol diet throughout the study, with a portion of the animals receiving feed supplemented with atorvastatin (44 mg/kg). The following treatment combinations were studied: - 1 β3-targeted paramagnetic nanoparticles without fumagillin and without atorvastatin (n = 5).
- 2 β3-targeted paramagnetic nanoparticles without fumagillin and with atorvastatin (n = 8).
- 3 β3-targeted paramagnetic nanoparticles with fumagillin (30 µg/kg body weight, baseline and week 4) and without atorvastatin (n = 5).
- 4 β3-targeted paramagnetic nanoparticles with fumagillin (30 µg/kg body weight, baseline) and with atorvastatin (n = 8).
- 5 β3-targeted paramagnetic nanoparticles with fumagillin (30 µg/kg body weight, baseline and week 4) and with atorvastatin (n = 8).
Aortic neovasculature signal was followed for 8 weeks with serial CMR molecular imaging at baseline and on weeks 1, 2, 4, 6, and 8. Before the last imaging session, blood was drawn to assess electrolytes, liver function, and hematology to assess the chronic effects of the high-lipid diet and experimental treatments. All samples were analyzed by the Washington University Department of Comparative Medicine.
Histology
A separate cohort of atherosclerotic animals (n = 7) was used for histological determination of β3-targeted nanoparticle binding. In 1 animal, β3-targeted rhodamine nanoparticles were injected and allowed to circulate for 3 h. Fifteen minutes before the animal was killed, fluorescein isothiocyanate (FITC)-labeled lectin was administered intra-arterially. Unbound nanoparticles and lectin were flushed from the vasculature by saline perfusion. The aorta was excised, frozen in optimal cutting temperature compound medium, sectioned, counterstained with 4',6-diamidino-2-phenylindole, and examined with fluorescence microscopy. Adjacent sections were stained with hematoxylin and eosin for light microscopy.
In the remaining animals, CMR molecular imaging of angiogenesis was performed with β3-targeted paramagnetic nanoparticles, followed by killing the animals for histological measurement of microvessel density. Formalin-fixed samples were paraffin embedded, sectioned, and stained for the expression of β3-integrin (LM-609, Chemicon International, Temecula, California) and platelet/endothelial cell adhesion molecule 1 (Chemicon International) (18). LM609-positive microvessel density was measured in digitized images from 3 independent full-transverse sections per animal.
MR molecular imaging of angiogenesis.
The β3-targeted paramagnetic nanoparticles (1 ml/kg) were injected into the marginal ear vein. Animals were imaged before and 3 h after injection with a high-resolution, 2-dimensional, T1-weighted, fat-suppressed turbo spin-echo sequence (250 x 250 µm resolution, 4-mm-thick slices, TR/TE 380/11 ms, 90° flip angle, SPIR fat suppression, turbo factor 4, 6 signal averages, 12.5 min scan time) using a clinical 1.5-T scanner (Philips Medical Systems, Andover, Massachusetts) and a quadrature birdcage coil. Three imaging stacks were acquired covering the descending thoracic aorta (10 slices), the transverse aorta (5 slices), and the ascending thoracic aorta (5 slices). Saturation bands were positioned superior and inferior to the imaging stacks to suppress signals from in-flowing blood.
CMR image analysis.
The CMR signal enhancement from the aortic wall was quantified using a custom semiautomated segmentation program previously described (17,18). Briefly, the aortic lumen was defined in each 2-dimensional image with a seeded region-growing algorithm. The aortic wall was defined by dilation of the luminal mask followed by an automated threshold to obtain a consistent and objective region of-interest encompassing the entire aortic wall (Fig. 1), which was evaluated by visual inspection. Image intensity was normalized across animals and time points to a fiduciary marker (test tube with 25 µmol/l gadolinium diethylene-triamine-pentaacetic acid in saline) placed within the field of view. The percentage enhancement in the CMR signal was calculated slice by slice in the 3-h post-injection images relative to the average pre-injection CMR signal, providing an unbiased integrated measurement of contrast enhancement in the aortic wall. The aortic contrast data represents the mean enhancement of all properly segmented slices.
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Figure 1 Segmentation of the Aortic Wall and Color-Coded Signal Enhancement Before and After Targeted Fumagillin Treatment
(Top) Black blood image of the thoracic aorta (arrow) and segmentation of the vessel wall (outlined in yellow) is shown for the week 0 image. The color-coded overlay of signal enhancement (%) shows patchy areas of high angiogenesis. On the week 1 image, the signal enhancement has clearly decreased due to the antiangiogenic effect of targeted fumagillin treatment. (Bottom) The level of signal enhancement gradually increases at weeks 2 and 3 after fumagillin treatment, until week 4, when the level of enhancement is practically identical to the week 0 image.
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Statistical analysis.
All data were analyzed using general linear models (i.e., regression, analysis of variance [ANOVA] and analysis of covariance), and group means for model effects were separated using least-significant differences (p < 0.05) (SAS Institute, Cary, North Carolina) and reported as mean ± standard error of the mean.
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Results
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Duration of antiangiogenic effect after treatment with vβ3-integrin targeted fumagillin nanoparticles.
At baseline, the average percentage signal enhancement from β3-targeted paramagnetic nanoparticles did not differ between treatment groups and ranged between 20% and 25% (Fig. 1). Consistent with previous reports, β3-targeted paramagnetic contrast was diffusely distributed across and within slices, and the enhancement calculated represented the average of all aortic voxels rather than a thresholded subset (17,18). Targeted characterization of β3-integrin expression revealed that early CMR neovascular signal enhancement did not differ between fat-fed animals switched to standard chow and those continuing on the cholesterol-enriched diet over the 4-week study, which reflects the marked serum cholesterol and fatty livers observed in all of the animals. For clarity of presentation, these treatment groups were pooled (i.e., control).
In the top panel of Figure 2, the overall effects of β3-targeted fumagillin nanoparticles on aortic neovascular contrast as a function of post-treatment diet are presented. At week 1, neovascular contrast among animals receiving β3-targeted fumagillin nanoparticles was decreased by approximately 50% to 75% relative to the control animals (p < 0.05). The decreased neovascular signal persisted through week 2. On week 3, aortic angiogenesis remained less than the control (p < 0.05) in both dietary groups, but the difference was smaller, indicating that the effect of fumagillin was waning. By week 4, there was no difference in the CMR neovasculature signal between control and fumagillin-treated rabbits, regardless of diet.
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Figure 2 Aortic Signal Enhancement up to 4 Weeks After Treatment With Targeted Fumagillin Nanoparticles
(Top) Serial imaging of angiogenesis in untreated (triangles) and fumagillin-treated animals remaining on high-cholesterol diet (squares) or switched to normal chow (circles). Fumagillin treatment at week 0 reduced angiogenesis compared with untreated control animals (*p < 0.05), but withdrawing the high-cholesterol feed had no effect. (Bottom) Aortic angiogenesis in rabbits without treatment (triangles), with 30 µg/kg fumagillin (circles), or with 90 µg/kg fumagillin (squares). Fumagillin treatment at 30 versus 90 µg/kg produced identical responses. *p < 0.05.
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No change (p > 0.05) in antiangiogenic response profile was found by increasing the fumagillin dose from 30 to 90 µg/ml over the 4-week period (Fig. 2). Although no difference in antiangiogenic response was appreciated between drug loading levels, the higher dose exhibited a trend toward longer effectiveness compared with its own signal enhancement at baseline.
Antiangiogenic synergism of β3-targeted fumagillin nanoparticles and atorvastatin.
Consistent with the earlier 4-week experiment, the CMR signal enhancement from β3-targeted paramagnetic nanoparticles averaged between 20% and 25% at baseline (Fig. 3). The neovascular contrast enhancement among hyperlipidemic control rabbits was constant over the 8-week study. Therapy with atorvastatin did not affect the CMR angiogenic signal at these time points. The top panel of Figure 3 shows that β3-targeted fumagillin nanoparticles elicited the same acute antiangiogenic response as observed previously in the present study and in our previous report (18). The first dosage of β3-targeted fumagillin nanoparticles decreased the angiogenic signal for 2 weeks followed by a return to the baseline level after 4 weeks. The second administration of β3-targeted fumagillin nanoparticles mirrored the antiangiogenic effects observed after the first dosage, suggesting that the antineovascular response was not influenced by the preceding drug treatment.
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Figure 3 Cardiac Magnetic Resonance Signal Enhancement up to 8 Weeks After Treatment With Targeted Fumagillin Nanoparticles With and Without Oral Atorvastatin
(Top) Cardiac magnetic resonance enhancement in untreated (triangles), atorvastatin-treated (circles) and fumagillin-treated animals (squares) during 8 weeks of follow-up imaging. Untreated and statin-treated animals showed a constant level of angiogenesis in the aortic wall. Animals treated with targeted fumagillin nanoparticles at 0 and 4 weeks showed decreased angiogenesis (*p < 0.05) after each dose, which returned to baseline levels within 4 weeks. (Bottom) Enhancement in rabbits receiving atorvastatin alone (triangles) or in conjunction with 1 (squares) or 2 (circles) doses of targeted fumagillin nanoparticles. The combination of 2 fumagillin doses and statin produced a sustained decrease in angiogenesis (*p < 0.05).
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The antiangiogenic effects of atorvastatin alone and in combination with β3-targeted fumagillin nanoparticles given once (baseline) or twice (baseline and week 4) are presented in the lower panel of Figure 3. Atorvastatin alone did not affect the CMR signal enhancement from the aortic neovasculature over the 8-week study. Administration of dietary atorvastatin and a single dose of β3-targeted fumagillin nanoparticles at baseline produced no change in the antiangiogenic response pattern. In addition, continued dietary atorvastatin following fumagillin treatment during the first 4-week period did not elicit an antiangiogenic effect during weeks 4 to 8.
Serial treatment with β3-targeted fumagillin nanoparticles at baseline and week 4 in conjunction with dietary atorvastatin resulted in the expected pharmacodynamic 4-week cyclic pattern observed previously (Fig. 2). However, the antiangiogenic response to the second dose of β3-targeted fumagillin nanoparticles was sustained by atorvastatin rather than returning to baseline levels. The CMR neovascular signals on weeks 6 and 8 after the second fumagillin treatment remained at less than one-half of the baseline contrast levels. Although atorvastatin alone exerted no significant effect on plaque angiogenesis in this short study, it is clear that dietary statin therapy can sustain the acute antineovascular benefits of β3-targeted fumagillin. The additive effects of fumagillin or statin alone were far less than the synergistic effect of both together. In the ANOVA, this was reflected by the interaction term (statin x fumagillin) p value of <0.0001, signifying a nonparallel response.
Clinical pathology.
Blood samples drawn at the end of the 8-week study were assessed for hematology, electrolytes, and liver function (Fig. 4) and compared with laboratory reference ranges published by the University of Minnesota Research Animal Resources program. Leukocyte, hemoglobin, hematocrit, and electrolyte (sodium, potassium, and chloride) values were within the normal range for rabbits and did not differ among treatment groups (p > 0.05). Platelet count averages were similar (p > 0.05) across treatments ( 370,000) and elevated somewhat compared with the reported normal upper limit for rabbits (270,000), which is considerably lower than the normal limit for other mammalian species.
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Figure 4 Liver Function Enzymes After 8 Weeks of Atorvastatin Treatment With or Without Targeted Fumagillin Nanoparticles
Blood samples drawn at the end of the 8-week study were assessed for liver function enzymes to assess the chronic effects of the high-lipid diet and experimental treatments. All values tended to be within normal ranges and identical for all treatment groups. Alkaline phosphate (AlkP), however, was elevated in all groups, and alanine aminotransferase (ALT) was increased in animals receiving 2 doses of targeted fumagillin nanoparticles and oral statin (Fum 2x + Statin) (*p < 0.05). AST = aspartate aminotransferase; Fum = fumagillin; GGT = gamma glutamyltransferase; NS = not significant.
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Alkaline phosphatase (AlkP) was 2- to 3-fold higher than the normal upper limit for all treatment groups, which was numerically highest in the control animals but not statistically different (p > 0.05). Gamma glutamyltransferase (GGT) was similar among treatment groups (p > 0.05), but like AlkP it was numerically highest in the control rabbits. Because a published normal range for GGT in rabbits was unavailable, the normal range for other nonhuman mammals was used. Aspartate aminotransferase (AST) was well within the normal limits and did not differ among treatment groups (p > 0.05). Alanine aminotransferase (ALT) was increased (p < 0.05) in rabbits receiving 2 doses of fumagillin and dietary statin compared with the normal range and the other treatment groups. Interestingly, the statin-only group also showed slightly elevated ALT compared with the control and fumagillin-alone groups. Although dietary statin combined with a single dose of β3-targeted fumagillin nanoparticles elevated ALT higher than either drug alone, the level was not beyond the normal range. Clinical pathology responses were uncorrelated with serum cholesterol level.
The increased AlkP and GGT values and the frank yellow discoloration of rabbit livers noted on necropsy suggested a marked fatty liver pathology without concomitant hepatobiliary disease. The increase in ALT in relation to AST also points to mild subclinical hepatic insult by statins alone, which are known to induce liver enzyme release, and in combination with the perfluorocarbon nanoparticles, which are predominantly cleared by the liver reticular endothelial cells (21).
Histology.
Fluorescence microscopy confirmed that β3-targeted rhodamine nanoparticles were distributed in the aortic adventia and were colocalized with FITC-lectin bound to vascular endothelium (Fig. 5). The β3-targeted rhodamine nanoparticles were not observed in the extravascular regions of the adventia or within plaque.
The microvascular density followed a logarithmic relationship (R2 = 0.84), with the CMR signal enhancement of β3-targeted paramagnetic nanoparticles (Fig. 6). Neovascular signal enhancement increased monotonically at higher advential microvessel counts and declined rapidly as neovessel counts decreased.
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Discussion
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In the present study, we demonstrated the concept of a prolonged antiangiogenic regimen with potential for clinical translation using a nanomedicine-based strategy, which offers CMR molecular imaging for patient stratification and monitoring and targeted drug delivery. Using CMR molecular imaging with β3-targeted paramagnetic nanoparticles, the pharmacodynamic antiangiogenic effectiveness of a single low dose of β3-targeted fumagillin nanoparticles was found to persist for 3 weeks regardless of drug loading, after which continued inflammation led to a recrudescence of the neovasculature to baseline levels. Atorvastatin alone did not induce neovascular changes detectable by CMR molecular imaging with β3-targeted paramagnetic nanoparticles over 8 weeks of study; however, the combination of targeted fumagillin nanoparticles and atorvastatin synergistically sustained the antiangiogenic effect. Microscopic studies corroborated the high correlation between CMR signal and neovessel counts and reconfirmed that the β3-targeted nanoparticles were constrained to the vasculature of the aortic adventia.
The integrin-targeted nanoparticles incorporated very low dosages of fumagillin, a mycotoxin produced by Aspergillus fumigatus that suppresses angiogenesis by inhibition of methionine aminopeptidase 2 (22). Methionine aminopeptidase 2 is responsible for cleavage of the NH2-terminal methionine residue from nascent proteins (23) and is up-regulated during cellular proliferation (24). TNP-470 is a water-soluble form of fumagillin which shares the active ovalicin core and analogously inhibits proliferating endothelial cells (i.e., angiogenesis) with little effect on nonendothelial cell types (25). TNP-470 has been studied in human clinical cancer trials, and anecdotal cases of disease remission, regression, or stabilization have been reported (26,27). Unfortunately, TNP-470 exhibited significant side effects at dosages required for therapeutic effects (60 to 100 mg/kg, serially), including sudden moderately severe symptoms of neurotoxicity. In the current nanoparticle application, the equivalent dosage of fumagillin (the parent compound) was 10,000-fold less than the total drug dose used in a prior murine apolipoprotein E–/– atherosclerotic model (28) yet still achieved remarkable end point efficacy. Similarly, the total gadolinium dose used in these experiments is about 100 times lower than that approved for clinical contrast imaging, which should diminish the potential for nephrogenic systemic fibrosis, which has been reported in patients with advanced renal disease or liver transplantation (29).
Ligand-directed perfluorocarbon nanoparticles are a theranostic platform technology targeted in this case to proliferating endothelial cells. The targeted vβ3-integrin is a heterodimeric transmembrane glycoprotein that is differentially up-regulated in proliferating versus quiescent endothelial cells but also expressed by numerous cell types prominently represented in atherosclerotic plaques, including endothelial cells (30,31), macrophages (32), platelets (33), lymphocytes (33), and smooth muscle cells (34). In this study, fluorescence microscopy demonstrated that perfluorocarbon nanoparticles (250 nm) were confined to endothelial targets within the advential microvasculature at the time of imaging.
The potential role of antiangiogenic therapy in the treatment of atherosclerosis has garnered increasing scientific discussion both for (15,35) and against (2,36) it. Although there is general agreement that angiogenesis is a prominent feature of vulnerable and ruptured plaques versus more stable fibrocalcific lesions, questions regarding the causative relationship and clinical relevance of antiangiogenic therapeutic strategies in cardiovascular disease have been raised. In the present work, we have shown how a theranostic nanomedicine approach can be interwoven with standard clinical practice to provide a sustained antiangiogenic regimen. Moreover, noninvasive CMR longitudinal monitoring of atherosclerotic disease with β3-targeted paramagnetic particles alone or enhanced with CMR intraplaque hemorrhage assessment (37) offers an attractive quantitative approach to continued medical management, particularly in asymptomatic patients.
Study limitations.
The utility of the New Zealand White atherosclerotic rabbit model derives from the development of an early-expanded vasa vasorum and neovasculature in response to the lipid-infiltrated aortic wall. Unfortunately, many features of human vulnerable plaque are lacking, including intraplaque hemorrhage, fibrous cap thinning, and plaque rupture. Therefore, important questions surrounding the hypothesis that antiangiogenic treatment will prevent or reduce the incidence of intraplaque hemorrhage as a result of neovessel pruning is poorly addressed in this animal model. Moreover, this model does not develop CMR-visible lesions, which precludes the use of CMR with endogenous contrast (37,38) for monitoring plaque size and/or composition, whereas other animal models involving balloon injury, de-endothelialization, and genetic modifications more closely mimic certain pathology features of vulnerable plaques, such as large lipid cores. None of the models compatible with nanoparticle research fully embrace the morphologic complexity and biochemical interplay of the human atherosclerotic plaque.
Additionally, hyperlipidemic rabbits suffer increased morbidity beyond 150 days on chronic cholesterol diet, which precluded determining the long-term duration of reduced angiogenesis achieved in the group receiving fumagillin (2x) and statin. Finally, one might reasonably speculate that a single dose of fumagillin following a month of statin treatment could yield a similar sustained reduction of atherosclerotic angiogenesis, which would be more desirable from patient safety, compliance, and health care cost perspectives.
Although we did not repeat competitive binding studies in these experiments due to logistic constraints, we demonstrated previously that these β3-targeted nanoparticles bind specifically to the neovascular endothelium with high affinity and avidity (17). The reproducibility of the present and previous CMR imaging results and the targeted antiangiogenic effects over multiple cohorts of rabbits, together with the "washout" studies showing recrudescence without continued therapy followed by efficacy upon retreatment, corroborate the robustness of this agent for diagnostic imaging and targeted drug delivery in vivo.
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Conclusions
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Others have postulated that antiangiogenesis therapy may normalize atherosclerotic plaque vasculature through neovascular pruning, which could diminish intraplaque hemorrhage frequency and promote plaque stabilization. The overarching hypothesis of the present studies was to determine whether the acute antiangiogenic effects of β3-targeted fumagillin nanoparticles could be incorporated into a clinically translatable regimen for testing the potential benefits of long-term antineovascular therapy. Cardiac magnetic resonance molecular imaging with β3-targeted paramagnetic nanoparticles was used to noninvasively demonstrate that a single minute dose of β3-targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks and that this effect was prolonged with the addition of atorvastatin. This theranostic nanomedicine approach could translate into a clinically relevant strategy to evaluate prolonged antiangiogenic treatment and atherosclerotic plaque stability.
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Acknowledgments
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The authors extend sincere appreciation to Grace Hu for analytical support, to Ralph Fuhrhop and Elizabeth Lacy for formulation chemistry, to John Allen and Cordelia Caradine for their assistance with the rabbit model, and to Michael Scott for program management support.
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Footnotes
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Supported in part by the National Cancer Institute, National Heart Lung and Blood Institute, and the National Institute for Biomedical Imaging and Bioengineering (HL-78631, HL-73646, N01-CO-37007, N01-CO-27031-16, CA-119342, and EB-01704) and by Philips Healthcare and Philips Research. Dr. Winter is a consultant to Kereos (St. Louis, Missouri). Dr. Caruthers is an employee of Philips Medical Systems (Andover, Massachusetts). Dr. Wickline receives equipment support from Philips Healthcare (Andover, Massachusetts). Drs. Wickline and Lanza are founders and stockholders of Kereos.
* Reprint requests and correspondence: Dr. Gregory M. Lanza, Washington University School of Medicine, 4320 Forest Park Avenue, Cortex Building, Suite 101, Campus Box 8125, St. Louis, Missouri 63108 (Email: greg{at}cvu.wustl.edu).
Manuscript received March 23, 2008;
revised manuscript received May 19, 2008,
accepted June 16, 2008.
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