Molecular Imaging of the Initial Inflammatory Response in Atherosclerosis
Implications for Early Detection of Disease
Background— We hypothesized that molecular imaging of endothelial cell adhesion molecule expression could noninvasively evaluate prelesion atherogenic phenotype.
Methods and Results— Mice deficient for the LDL-receptor and the Apobec-1 editing peptide (DKO mice) were studied as an age-dependent model of atherosclerosis. At 10, 20, and 40 weeks of age, ultrasound molecular imaging of the proximal thoracic aorta was performed with contrast agents targeted to P-selectin and VCAM-1. Atherosclerotic lesion severity and content were assessed by ultrahigh frequency ultrasound, histology, and immunohistochemistry. In wild-type mice at all ages, there was neither aortic thickening nor targeted tracer signal enhancement. In DKO mice, lesions progressed from sparse mild intimal thickening at 10 weeks to widespread severe lesions with luminal encroachment at 40 weeks. Molecular imaging for P-selectin and VCAM-1 demonstrated selective signal enhancement (P<0.01 versus nontargeted agent) at all ages for DKO mice. P-selectin and VCAM-1 signal in DKO mice were greater by 3-fold at 10 weeks, 4- to 6-fold at 20 weeks, and 9- to 10-fold at 40 weeks compared to wild-type mice. En face microscopy demonstrated preferential attachment of targeted microbubbles to regions of lesion formation.
Conclusions— Noninvasive ultrasound molecular imaging of endothelial activation can detect lesion-prone vascular phenotype before the appearance of obstructive atherosclerotic lesions.
The inflammatory response plays an important role in the initiation and progression of atherosclerosis and the susceptibility to acute ischemic syndromes. Novel methods for detecting vascular inflammation are being developed to better ascertain risk for adverse events. One strategy is to directly visualize the molecular or cellular components of the immune response with targeted imaging probes. This approach could also potentially be used for early identification of individuals predisposed to severe atherosclerosis by detecting phenotypic changes that occur decades before clinical manifestations arise.1
The aim of this study was to test whether the early and inciting events of atherosclerosis can be detected in vivo by molecular imaging. The targets for this study, VCAM-1 and P-selectin, are key endothelial cell adhesion molecules that regulate leukocyte trafficking in atherosclerosis.2–5 Surface expression of both of these molecules occurs in response to oxidized LDL cholesterol and are present early at lesion-prone sites.2–10 Most targeted imaging technologies have been tested in models where plaque formation and inflammatory cell influx are advanced. However, ex vivo microscopy studies have demonstrated that imaging probes targeted to VCAM-1 or P-selectin accumulate in early atherosclerotic lesions.11,12 In this study, we hypothesized that molecular imaging in vivo could detect upregulation of adhesion molecules before the development of advanced atherosclerotic lesions. To test our hypothesis, targeted imaging and histomorphometric analysis were performed at various ages in mice with a genetic deletion of both the LDL receptor and the apolipoprotein (Apo) mRNA editing protein Apobec-1 that is responsible for preferential transcription of ApoB-48 over ApoB-100 in mice.13,14 These double knockout (DKO) mice have high circulating LDL cholesterol with full-length ApoB-100 and develop a predictable age-dependent course of atherosclerosis in the proximal aorta and all branch points while on a chow diet.14
The study was approved by the Animal Care and Use Committee of the Oregon Health & Science University. Control wild-type C57Bl/6 mice and 33 DKO mice with gene-targeted deletion of the LDL receptor and Apobec-1 on a C57Bl/6 background underwent imaging studies at 10, 20, or 40 weeks of age (n=7 to 10 for each strain at each age) (Genentech, San Francisco, Calif.). Approximately half of the mice were euthanized after either the 10 or 20 weeks imaging study for aortic histomorphometry and immunohistochemistry for adhesion molecule expression. Total lesion area from en face oil red O staining of aortic whole mounts was performed in an additional 15 DKO mice (n=5 for each age). Spatial characterization of adhesion for fluorescently-labeled targeted microbubbles was assessed from ex vivo microscopy of the aortic arch in DKO mice.
Biotinylated lipid-shelled decafluorobutane microbubbles were prepared by sonication of a gas-saturated aqueous suspension of distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-PEG(2000)biotin (Avanti Polar Lipids). Rat antimouse monoclonal IgG1 against either VCAM-1 (MK 2.7) or P-selectin (RB40.34), or isotype control antibody (R3–34, Pharmingen Inc) were conjugated to the surface of microbubbles as previously described to produce control, P-selectin-targeted, or VCAM-1-targeted microbubbles.15,16 The control preparation has been shown to result in minimal nonspecific attachment in vivo and in flow chamber models with cultured endothelial cells.15,16 Microbubble size distribution and concentration were measured by electrozone sensing (Multisizer III, Beckman Coulter). Size distribution was not different between agents nor was the surface density of ligand determined by flow cytometry (saturating concentration at 20 μg per 1×108 microbubbles for all three agents).15
Targeted Imaging of VCAM-1 and P-Selectin
Mice were anesthetized with inhaled isoflurane. A jugular vein was cannulated using sterile technique. Contrast-enhanced ultrasound (CEU) of the aorta was performed with a high-frequency linear-array probe (Sequoia, Siemens Medical Systems) fixed in place by railed gantry system. The ascending aorta, proximal aortic arch, and proximal brachiocephalic trunk were imaged in long axis using a right parasternal imaging approach. For CEU, the nonlinear fundamental signal component for microbubbles was detected with a multipulse protocol (Contrast Pulse Sequencing) at a transmission frequency of 7 MHz, a mechanical index of 1.1 to 1.2, and a dynamic range of 40 dB. Gain settings were adjusted to levels just below visible noise speckle and held constant. These settings provide a linear and reproducible relationship between contrast concentration and intensity obtained in the study. Images were acquired 10 minutes after the intravenous injection of targeted or control microbubble agents (1×106 per injection, performed in random order). After several frames were acquired at a pulsing interval (PI) of 1s, the mechanic index was briefly increased to 1.5 to 1.7 to completely null microbubble signal then several frames were acquired at a PI of 5 s to measure the signal from freely circulating microbubbles. Video intensities were transformed from a log-compressed to linear scale using the log function for the ultrasound system. Several postdestruction frames were averaged and digitally subtracted from the first two predestruction frames to derive signal from retained microbubbles only (see supplemental Figure A, available online at http://atvb.ahajournals.org).15 Intensity was measured from a region-of-interest placed around the ascending aorta and aortic arch and extending into the origin of the brachiocephalic artery. Region selection was guided by fundamental frequency imaging at 14 MHz acquired at the end of each CEU acquisition. In vitro water bath experiments were performed with the in vivo settings using incremental microbubble concentrations in a 2 L water bath with a stir bar. A total of 5 frames at a PI of 1 s were analyzed and averaged.
Plaque Morphometry by Ultrasound
High-resolution imaging of the aortic arch was performed with a high-frequency (40MHz) imaging system with an integrated physiology monitoring platform (Vevo 770, VisualSonics Inc). Measurements were made at end-diastole by gating individual line acquisition to the ECG (frame rate of ≈1000 Hz). Plaque burden was assessed by measuring vessel wall thickness at the lesser curvature of the arch and the origin of the brachiocephalic artery (intraobserver variability 7±3%, interobserver variability <15%). Left ventricular fractional shortening, averaged for the 2 short-axis diameters, was assessed in the parasternal short axis plane at the midpapillary muscle level. Centerline peak systolic velocity was measured with pulsed-wave Doppler with the sample volume placed in the aortic arch just distal to the brachiocephalic artery.
Ex Vivo Imaging of Microbubble Adhesion
In anesthetized DKO mice at 20 or 40 weeks of age (n=3 for each), P-selectin–, or VCAM-1-targeted microbubbles (1×106) fluorescently labeled with the dialkylcarbocyanine fluorochrome DiI were injected via a jugular vein. Control microbubbles (1×106) labeled with DiO were injected simultaneously. After 3 minutes of circulation time, the blood volume of the mouse was removed by infusion of 8 mL isothermic heparinized saline at physiological pressure through a left ventricular puncture. The thoracic aorta was removed, incised longitudinally, and pinned to allow en face observation of the endothelial surface. Microbubble attachment relative to plaque development was characterized using fluorescent epi-illumination (excitation filter 460 to 490 nm and 530 to 560 nm).
For histology, perfusion fixation was performed and short axis sections of the mid ascending aorta and proximal arch just before the brachiocephalic artery were snap-frozen providing 2 separate regions for each subject. Movat pentachrome stain was performed for assessment of plaque area measured by the tissue area within the internal elastic lamina. For immunohistology of VCAM-1 expression, a goat polyclonal antibody for VCAM-1 (sc1504, Santa Cruz Biotechnology Inc) with secondary peroxidase staining (ABC Vectastain Elite, Vector Laboratories) was used. Because P-selectin is prestored within endothelial cells, surface expression was evaluated by in vivo labeling. Streptavidin Qdot nanocrystals (Invitrogen, 605 nm peak emission spectrum) bearing Rb40.34 were injected intravenously (20 μL) in 2 wild-type and 4 DKO mice at 10 or 40 weeks of age. The blood volume was rinsed by left ventricle puncture and the aorta was dissected free, incised longitudinally, and observed under fluorescent illumination. Determination of en face plaque area for the thoracic aorta was determined from methanol-fixed (78%) whole mounts stained with 0.15% oil red O (Sigma Aldrich).
Data were analyzed on SAS (version 9.1) or RS/1 (version 6.0.1, Domain Manufacturing Corp). Data were analyzed with 1-way ANOVA for normally distributed variables with post hoc testing of individual comparisons with paired t test, or by the Wilcoxon rank-sum test for medians. Bonferroni correction was applied for multiple comparisons. A Spearman rank correlation test was used to assess the relationship between age and histology data.
Cardiac Performance and Aortic Flow Velocities
On echocardiography left ventricular fractional shortening, aortic internal diameter, and aortic peak systolic flow velocities were similar for DKO and wild-type mice at each age (Table). These data indicate that hemodynamic and shear rates that can influence microbubble adhesion in the aorta were similar between groups.
Vessel Morphometry and Histology
Oil red O staining of aortic whole mounts detected an age-dependent increase in plaque en face area in DKO mice, whereas staining was essentially absent in wild-type animals (Figure 1A). On histology with Movat staining (Figure 1B), there was no intimal thickening in wild-type mice at any age. In DKO mice, at 10 weeks of age there were regions of mild intimal thickening and occasional monocyte adhesion to the endothelial surface seen in several animals, although the majority of sections had no significant abnormalities. At 20 weeks of age, small but discrete fibrous or fibrofatty lesions were frequently detected and were often associated with disruption of the elastic lamina. These lesions rarely encroached into the lumen area. All sections from 40-week-old DKO mice demonstrated severe lipid-rich lesions with abundant inflammatory cells and regions of necrosis. These lesions tended to protrude well into the aortic lumen.
Vessel wall thickness measured by ultrahigh frequency ultrasound of was ≤60 μm in wild-type mice and did not change with age (Figure 2, supplemental Video I). In DKO mice, wall thickness at 10 weeks of age was similar to wild-type mice. At 20 weeks, there was a small increase in arterial wall thickness in DKO mice that did not reach statistical significance, indicating that the small lesions seen on histology could not be reliably detected by high-frequency ultrasound. At 40 weeks of age, ultrasound detected marked arterial thickening of the lesser curvature of the aorta and focal plaque formation at the proximal brachiocephalic artery (Figure 2, supplemental Video II).
On immunohistochemistry for VCAM-1, there was minimal staining in wild-type mice at all ages. In DKO mice, VCAM-1 was present on the intimal surface at 10, 20, and 40 weeks of age (Figure 3A through 3D). At 40 weeks of age, VCAM-1 was particularly intense on the endothelium at shoulder regions and within the complex lesions containing immune cells. Aortic whole mounts with in vivo labeling with P-selectin-targeted Q-dots demonstrated endothelial surface expression of P-selectin only in DKO mice, particularly at sites of lesion formation (supplemental Figure B).
Targeted Imaging of Adhesion Molecule Expression
Fluorescent microscopy of aortic whole mounts revealed attachment of both P-selectin and VCAM-1-targeted microbubbles diffusely on the surface of regions of early plaque formation in 20 weeks DKO mice (Figure 3E and 3F, additional images available in the online supplemental data). Adhesion was particularly dense along the proximal margins of the plaques. Adhesion to normal regions was sparse except at the origin of intercostal branches in the descending portion of the thoracic aorta. In 40-week DKO mice, targeted microbubble adhesion was again more frequent in regions of plaque formation, although preferential adhesion at the plaque margins was much less prominent than at 20 weeks. Irrespective of age, targeted microbubble adhesion was most dense in lesions at the origin of the brachiocephalic artery. Control microbubble attachment was rare and not related to any particular plaque topography.
CEU molecular imaging signal in the ascending aorta and arch of wild-type mice was similar between control microbubbles and targeted microbubbles (Figure 4A). In DKO mice there was selective signal enhancement for P-selectin and VCAM-1-targeted microbubbles compared to control microbubbles at 10, 20, and 40 weeks of age. Signal from stationary targeted microbubbles occurred at both the specular edges of the vessel and “within the lumen” due to volume averaging of the entire mouse aorta within the elevational profile of the beam.16 P-selectin and VCAM-1 signal in DKO mice was greater than wild-type controls at all time points. The ratio of targeted signal in DKO mice versus control mice increased with age (approximately 3-fold greater at 10 weeks, 4- to 6-fold greater at 20 weeks, and 9- to 10-fold at 40 weeks). A diffuse pattern of enhancement throughout the aortic arch was seen in 40 weeks DKO mice (illustrated in Figure 4). Signal enhancement at the early time intervals (10 and 20 weeks) was often focal, illustrated in Figure 5, which also demonstrates how spatial localization was performed. A linear relation between the microbubble concentration and CEU intensity was found from in vitro water bath experiments (Figure 6).
Upregulation and surface expression of vascular endothelial cell adhesion molecules are early events in atherogenesis. Results from this study performed in a reproducible age-dependent murine model of aortic atherosclerosis indicate that molecular imaging of pathogenic endothelial cell adhesion molecules such as VCAM-1 and p-selectin can detect atheresclerotic vascular phenotype before the development of advanced lesions.
Over the past decade, there has been increasing interest in the development of molecular imaging methods to assess the inflammatory response in atherosclerotic disease. Probe design has been governed by the intended clinical use. For example, the differentiation of high-risk “vulnerable” disease from more stable disease requires a technique that can detect the tempestuous late-stage events that contribute to plaque rupture or erosion. These events include macrophage infiltration, endothelial cell activation, protease activity, chemokine production, thinned fibrous cap, and increased metabolic activity. The aim of this study was to determine whether a molecular imaging approach could unmask early pathophysiologic changes and reveal risk for future development of severe disease. The scope of potential targets that can identify very early atherosclerosis is narrower than those for high-risk late-stage disease. One strategy has been to image the accumulation of oxidized lipoproteins in the vessel wall.17,18 In this study, our approach was to image the secondary inflammatory responses that occur in response to oxidized lipids by targeting microbubbles to adhesion molecules that are preferentially expressed at disease-prone regions.2–10
Interactions between P-selectin on the endothelial cell surface and modified glycoprotein counterligands on leukocytes mediates rolling and activation of leukocytes in postcapillary venules.19,20 These events are requisite for firm arrest and transmigration. Slow rolling and firm adhesion are, in part, mediated by interaction between leukocyte VLA-4 (α4β1) and VCAM-1 on the endothelial surface. The selection of P-selectin and VCAM-1 as endothelial markers of early disease was based not solely on their importance in the inflammatory response. These molecules play a critical role in leukocyte arrest in high-shear stress vessels such as the mouse aorta,21 and participate in the early stages of atherogenesis.4,5
It has been previously shown that expression of endothelial cell adhesion molecules such as VCAM-1 and P-selectin in advanced atherosclerotic disease can be imaged in vivo with ultrasound or MRI targeted imaging probes.11,12,16,22,23 VCAM-1 signal intensity on CEU has been shown to correlate with the severity of diet-modulated vascular inflammation in late stage ApoE−/− mice.16 Feasibility for detecting disease at an early stage has been suggested by ex vivo optical imaging of aortas from ApoE−/− mice where accumulation of VCAM-1-targeted optical probes can be observed at the site nonobstructive atherosclerotic lesions.11,12
In the present study we show for the first time that it is possible to noninvasively image vascular inflammation in atherosclerosis at the onset of inflammatory cell entry into the vessel wall before the development of intimal xanthomas (fatty streaks). It was interesting to note that targeted signal enhancement in DKO mice was relatively consistent between ages, which could reflect a constant pattern of endothelial expression, despite and increase in intraplaque VCAM-1 signal exemplified in Figure 3. However, signal enhancement for microbubbles targeted to P-selectin and VCAM-1 in DKO relative to wild-type mice did increase with age, possibly reflecting a limitation in using a fixed dose of contrast agent rather than indexed to weight. The age-related increase in en face plaque area and cross-sectional plaque area on Movat staining outpaced the age-related increase in targeted signal for DKO versus wild type animals. Based on the spatial pattern of microbubble attachment, we believe that minimal increase in signal despite increasing plaque area could also reflect the preferential expression of adhesion molecules such as VCAM-1 that occurs at the margins rather than the central “dome” of plaques.6,7
We believe that the detection of very early immune responses is an ideal application for molecular imaging with a CEU approach. Because early identification of proatherogenic vascular phenotype is likely to be used as a screening tool, the brevity of CEU molecular imaging protocols and availability of ultrasound in the outpatient setting are practical advantages. Transition to humans will be contingent on development of probes suitable for human use in terms of conjugation chemistry and ligand composition such as peptides that have been discovered through phage-display screening.23
There are several limitations of this study that deserve attention. We cannot yet draw any conclusions regarding whether disease management can be improved by molecular imaging of the early inflammatory response. Although ultrahigh-frequency imaging was able to evaluate focal aortic thickening, the lower-frequency contrast-specific imaging method did not provide adequate spatial resolution to colocalize signal enhancement with degree of thickening. This issue is related to the small scale of the animal model used and the phenomenon of volume averaging that occurs when the full dimension of the mouse aorta fits within the beam elevation.16 It is likely that high-frequency ultrasound measurements overestimated the relative degree of aortic thickening in 40-week DKO mice compared to controls, largely because the axial resolution (approximately 50 to 70 μm) precluded true representation of normal wall dimensions. The data do, however, support the notion that molecular imaging can detect the molecular signature of disease before even the most sensitive of techniques for evaluating morphology. We also did not evaluate the relative contribution of platelets to p-selectin signal enhancement,24 although histology studies did not show evidence of significant platelet thrombi.
In conclusion, we show that CEU molecular imaging can be used to noninvasively detect the expression of cell adhesion molecules that are involved in the early pathogenesis of atherosclerosis. These inflammatory changes can be imaged noninvasively before the development of advanced lesions. This method could in the future be useful for early risk stratification in atherosclerosis.
Sources of Funding
These studies were supported by grants R01-HL-074443, R01-HL-078610, and R01-DK-063508 to Dr Lindner from the National Institutes of Health, and from a grant from Genentech Inc. Dr Kaufmann is supported by grant SNF 32323B_123919/1 from the Swiss National Science Foundation and a grant from the Lichtenstein Foundation. Dr Carr is supported by a Postdoctoral Fellowship Grant from the Pacific Mountain Affiliate of the American Heart Association. Dr Chadderdon is supported by a Fellow to Faculty Transition Award from the American Heart Association.
Dr Lindner serves on the Scientific Advisory Board for VisualSonics Inc.
Received May 7, 2009; accepted October 6, 2009.
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