Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction

Abstract

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.

Figures

Figure 1

Structure of 111In-RP748, a quinolone targeted at αvβ3 integrin (A), and control compound (B).

Figure 2

Immunohistochemical analysis in chronic rat model. Example of lectin, αv, and β3 staining is shown for normal and infarct region (A) in rats 2 weeks following infarction. The infarct region demonstrated an increase in capillary density and arterioles, as well as increased staining for αv and β3. This was confirmed by quantitative analysis (B). *P < 0.05 vs. normal.

Figure 3

γ-Well counting of myocardial radiotracer activity in relationship to 201Tl uptake in the chronic rat model. Data are shown for rats injected with either 111In-RP748 (RP748) or control compound. Decreased myocardial 201Tl uptake was consistently observed in the anterolateral wall, as shown in representative myocardial count profiles (A). Cntrl, control; ant, anterior; sept, septal; post, posterior; lat, lateral. Uptake of 111In-RP748 was highest in infarcted regions with reduced 201Tl retention. In contrast, myocardial uptake of the control compound tracked 201Tl perfusion. On average the relative myocardial retention of 111In-RP748 in the post-ischemic and infarcted regions was nearly twice that in regions with normal 201Tl perfusion; however, no selective retention of the nonspecific control compound was observed (B). *P < 0.05 vs. 81_120%; #P < 0.05 vs. control.

Figure 4

Immunohistochemical analysis in chronic canine model. Masson’s trichrome staining demonstrated increased vascular density in the fibrotic central infarct region, which was confirmed by lectin staining. The infarct region demonstrated increased staining for αvβ3 using the LM609 Ab (A). LM609 staining localized to the endothelial cells of capillaries and endothelial cells, as well as smooth muscle cells of small arterioles within the infarct region. Very little LM609 staining was in seen in remote noninfarcted regions. These regional differences in staining were confirmed by quantitative analysis (B). *P < 0.05 vs. normal.

Figure 5

Well counting of myocardial radiotracer activity in relationship to 99mTc-sestamibi perfusion in chronic canine infarct model. Ex vivo 99mTc-sestamibi perfusion (left) and 111In-RP748 (right) images of representative myocardial slices from an infarcted dog, 3 wks after reperfusion, with the corresponding TTC-stained section (middle) (A). Corresponding circumferential 99mTc-sestamibi (MIBI) and 111In-RP748 count profiles are shown for both endocardial (ENDO) and epicardial (EPI) segments. Focal uptake of 111In-RP748 is seen in infarct region on ex vivo images, and is confirmed by γ-well counting. Myocardial segments from all dogs were segregated into four categories based on relative 99mTc-sestamibi perfusion (percentage nonischemic). Regional myocardial 111In-RP748 activity (percentage nonischemic) was significantly increased 3 wks after reperfusion in the ischemic regions of all dogs (B). #P < 0.05 vs. 81_120.

Figure 6

In vivo and ex vivo 111In-RP748 and 99mTc-sestamibi (99mTc-MIBI) images from dogs with chronic infarction. Serial in vivo 111In-RP748 SPECT short axis, vertical long axis (VLA), and horizontal long axis (HLA) images in a dog 3 wks after LAD infarction at 20 min and 75 min after injection in standard format (A). 111In-RP748 SPECT images were registered with 99mTc-MIBI perfusion images (third row). The 75-min 111In-RP748 SPECT images were colored red and fused with 99mTc-MIBI images (green) to better demonstrate localization of 111In-RP748 activity within the heart (color fusion, bottom row). Right ventricular (RV) and left ventricular (LV) blood pool activity is seen at 20 min. White arrows indicate region of increased 111In-RP748 uptake in anterior wall. This corresponds to the anteroapical 99mTc-sestamibi perfusion defect (yellow arrows). Sequential 99mTc-sestamibi (top row) and 111In-RP748 in vivo SPECT HLA images at 90 min after injection (middle row) from a dog at 8 h (acute), 1 wk, and 3 wks after LAD infarction (B). Increased myocardial 111In-RP748 uptake is seen in the anteroapical wall at all three time points. Color fusion 99mTc-MIBI (green) and 111In-RP748 (red) images (bottom row) demonstrate 111In-RP748 uptake within 99mTc-MIBI perfusion defect. Ex vivo 99mTc-sestamibi (left) and 111In-RP748 (center) images of myocardial slices from a dog 3 wks after LAD occlusion, with color fusion image on the right (C). Short axis slices are in the standard orientation. Yellow arrows indicate anterior location of nontransmural perfusion defect region; white arrows indicate corresponding area of increased 111In-RP748 uptake.

Figure 7

Evaluation of αvβ3 integrin localization in cultured canine endothelial cells. LM609, an established mAb of αvβ3 integrin, is localized at cell contact points (left). A cy3-labeled analogue of 111In-RP748 (TA145) also localized to αvβ3 at focal cellular contact points in a distribution similar to LM609 (middle). No focal cellular uptake was seen with the isotype-matched negative control Ab (MOPC21, right).