Abstract
Rationale and ObjectiveVascular cell adhesion molecule-1 (VCAM-1) is upregulated in ischemia reperfusion injury (IRI), persisting after restoration of blood flow. We hypothesized that microparticles of iron oxide targeting VCAM-1 (VCAM-MPIO) would depict “ischemic memory” and enable in vivo assessment of VCAM-1 expression.Methodology and FindingsMice subject to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO (4.5 mg iron/kg body weight). Contrast agent bound rapidly (<30 minutes) in IRI-kidneys and appeared as intensely low signal areas by MRI in vivo. Automated segmentation and quantification yielded MPIO contrast volumes of 5991±354×106 µm3 in IRI vs. 87±7×106 µm3 in kidneys with no surgical intervention (P<0.001); 90±8×106 µm3 in IRI kidneys exposed to control (IgG-MPIO) and 625±80×106 µm3, in IRI kidneys pre-treated with a blocking dose of VCAM-1 antibody (P<0.001). In keeping with quantitative MRI data, VCAM-1 mRNA expression in IRI was 65-fold higher than in kidneys without surgical intervention (3.06±0.63 vs. 0.05±0.02, P<0.001). Indeed VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R2 = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. Serial imaging showed VCAM-MPIO accumulation at target within 30 minutes, persisting for ≥90 minutes, while unbound VCAM-MPIO was cleared rapidly from blood, with sequestration by mac-3 positive Kupffer cells in the liver and monocyte/macrophages in the spleen.Conclusions(1) VCAM-MPIO detected VCAM-1 expression and defined its 3-dimensional distribution, revealing “ischemic memory” in renal IRI; (2) automated volumetric quantification of VCAM-MPIO accurately reflected tissue levels of VCAM-1 mRNA; and (3) VCAM-MPIO bound rapidly to target with active sequestration of unbound MPIO in the liver and spleen.
Highlights
In kidneys subjected to Ischemia-reperfusion injury (IRI), VCAM-microparticles of iron oxide (MPIO) caused a marked contrast effect that was evident as areas of low-signal intensity in both the renal cortex and medulla
To determine the optimal time point for imaging, we undertook serial magnetic resonance imaging (MRI) at 30 minute intervals, demonstrating that contrast effects were clearly apparent by 30 minutes and maximal by 60 minutes with persistent specific contrast in the kidneys at 90 minutes (Figure 2A)
Microparticles of iron oxide were rapidly cleared by the liver and spleen, as indicated by reduction in signal to noise ratio in those organs within 30 minutes (Figure 2B)
Summary
Ischemia-reperfusion injury (IRI) is an important pathological process in acute vascular syndromes including myocardial infarction,[1,2,3] stroke,[4,5] cardiac surgery[2,6] and organ transplantation.[7,8] A key feature of IRI is activation of inflammatory pathways, including the endothelial upregulation of adhesion molecules that mediate leukocyte slowing, rolling, and firm adhesion to the vessel wall.[9,10,11,12,13] Since these adhesion molecules persist on the vascular endothelial surface even after ischemia itself has resolved, their identification could represent a functional imprint or ‘memory’ of the prior ischemic insult.[14]. The ability to identify such an imprint non-invasively with magnetic resonance imaging (MRI) could provide more precise and rapid diagnosis and, potentially, guide targeted interventions. VCAM-1 and its ligand, a4b1 integrin ( called very late antigen-4, VLA-4), are important mediators of leukocyte recruitment and inflammation, including in IRI.[10] We have recently developed a targeted contrast agent for magnetic resonance molecular imaging. This agent, comprising antibody-conjugated microparticles of iron oxide (MPIO), has shown upregulation of VCAM-1 in a mouse model of cerebral inflammation, artificially induced by direct injection of interleukin-1.[15]
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