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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2005 as doi:10.1096/fj.05-4130fje. |
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vß3-targeted detection of arteriopathy in transplanted human coronary arteries: an autoradiographic study
,
,,,,,
,,1
* Raymond and Beverly Sackler Cardiovascular Molecular Imaging Laboratory, Section of Cardiovascular Medicine and
Section of Cardiothoracic Surgery,
Interdepartmental Program in vascular biology and transplantation, Yale University School of Medicine, New Haven, Connecticut, USA;
VA Connecticut Healthcare System, West Haven, Connecticut, USA; and
|| Bristol-Myers Squibb Medical Imaging, North Billerica, Massachusetts, USA
1 Correspondence: VA Connecticut Healthcare System, 950 Campbell Ave., 111B, West Haven, Connecticut 06516, USA. E-mail: mehran.sadeghi{at}yale.edu
SPECIFIC AIMS
Pathological remodeling is a common feature of a broad spectrum of vascular diseases. Graft arteriopathy (GA), the prototypic example of immune-mediated vascular remodeling, is responsible for the majority of cases of late graft loss in solid organ transplantation. There is currently no noninvasive imaging modality for early detection of GA. The development of a noninvasive imaging modality for early detection of GA could advance our understanding of basic pathophysiology and improve care of patients with cardiac transplantation. Furthermore, it could have major implications for other disorders associated with pathological remodeling.
The hallmark of GA is diffuse vascular remodeling associated with a flow-limiting, concentric neointima. The study of human GA has been limited by the difficulty of evaluating transplanted arteries in vivo and the lack of appropriate animal models. A recently described model of human artery transplantation onto the abdominal aorta of severe combined immunodeficiency (SCID)/beige mice, followed by allogeneic peripheral mononuclear cell (PBMC) reconstitution, mimics many pathological features of GA, including outward remodeling and neointima formation.
Vascular cell proliferation is a crucial step in the development of GA. Targeting cell proliferation may provide an opportunity to image the pathogenic process in vivo. The integrin 
vß3 is highly expressed on smooth muscle cells (SMC) and endothelial cells (EC). Its expression and activation play a key role in vascular cell proliferation and migration. RP748 is a novel 111In-labeled tracer with preferential binding to the activated conformer of vß3 integrin. We hypothesized that GA is associated with early up-regulation (and activation) of vß3 integrin and that the GA-associated proliferative process may be tracked by targeting (activated) vß3 integrin in vivo. Using the chimeric human/mouse model of GA, we demonstrate that vß3 is up-regulated in early GA, vß3 expression and cell proliferation follow a similar temporal pattern and in vivo uptake of RP748 tracks cell proliferation in GA. We conclude that RP748 may be used to detect the proliferative process in arteriopathy.
PRINCIPAL FINDINGS
1. The chimeric human/SCID mouse model of GA
GA was studied in a novel chimeric human/mouse model in which a segment of human coronary artery is transplanted to a SCID/beige mouse host followed by allogeneic PBMC reconstitution. There was considerable vascular remodeling, characterized by the formation of a thick neointima over a period of 4 wk after PBMC reconstitution. The ratio of neointima to media area increased from 0.3 ± 0.1 in the absence of PBMC (0 wk) to 1.0 ± 0.1 and 2.0 ± 0.3 at 2 and 4 wk after PBMC reconstitution (P<0.01 0 wk vs. 4 wk, and <0.05 2 wk vs. 4 wk, n=4).
2. vß3 integrin and cell proliferation in the chimeric model of human GA
The temporal pattern of vß3 integrin expression in coronary artery transplants was evaluated by IF staining. Nontransplanted human arteries or arteries from transplanted animals without PBMC reconstitution stained weakly for vß3, and the staining was predominantly limited to the endothelium (Fig. 1
a, b). By 2 wk after PBMC reconstitution, vß3 integrin was markedly up-regulated in the media of transplanted arteries (Fig. 1c)
. By 4 wk the expression declined toward baseline levels (Fig. 1d
).
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vß3 integrin is involved in vascular cell proliferation and migration. Therefore, we quantified vascular cell proliferation at different stages of GA by Ki67 staining. Little proliferation could be detected in nontransplanted arteries or those transplanted for 5 wk without PBMC reconstitution. Cell proliferation was maximal at 2 wk (proliferation index 2.1±1.0, 8.0±1.3.0, and 4.8±1.0, respectively, in no PBMC, 2 wk PBMC and 4 wk PBMC, P<0.01 for control vs. 2 wk, other differences nonsignificant n=5). As such, the temporal pattern of vß3 expression paralleled that of cell proliferation in GA.
3. RP748 uptake in GA in vivo tracks cell proliferation
To target vß3 integrin in vivo, RP748 (7.4 MBq) was injected to chimeric mice at various times after PBMC reconstitution. Animals were killed after 6 h and tracer uptake was quantified by autoradiography. RP748 uptake in the arterial transplant significantly increased by 2 wk after PBMC reconstitution and decreased toward control by 4 wk (relative uptake 1.2±0.0, 2.5±0.3, and 1.8±0.1, respectively, in the absence of PBMC and 2 and 4 wk after PBMC reconstitution, n=5, P<0.01 control vs. 2 wk, P<0.05 2 wk vs. 4 wk, and P<0.05 control vs. 4 wk. Other differences nonsignificant) (Fig. 2
A, B). As such, the temporal pattern of RP748 uptake in vivo closely tracked that of cell proliferation (Fig. 2C
).
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4. Specificity of RP748 uptake
To address the specificity of RP748 uptake in grafted arteries, chimeric SCID mice at 2 wk after PBMC reconstitution (corresponding to maximal RP748 uptake) were pretreated with excess nonlabeled tracer (TA138) prior to RP748 administration. Fifty-fold excess TA138 significantly inhibited RP748 uptake (relative uptake 1.3±0.0 in the presence of TA138, n=3, vs. 2.5±0.3 in its absence, n=5, P<0.01), demonstrating specificity of tracer uptake.
CONCLUSIONS AND SIGNIFICANCE
In this series of experiments we use a novel chimeric human/mouse model of GA to demonstrate that 1) GA is associated with early expression (and activation) of vß3 integrin, 2) the temporal pattern of vß3 expression parallels that of vascular cell proliferation, and 3) by targeting activated vß3 integrin in transplanted arteries, RP748 uptake can track the proliferative process.
GA lesions consist mainly of SMC, extracellular matrix, and infiltrating mononuclear cells. In response to immune injury, medial SMC proliferate and migrate to form the neointima. The vß3 integrin has been shown to dominate integrin-matrix interactions required for postinjury SMC migration. However, the extent of vß3 involvement in the development of GA has only been assessed in specific, limited situations. We assessed the temporal pattern of vß3 expression in an animal model of immune-mediated arteriopathy. vß3 is up-regulated early after allogeneic stimulation and the levels decline in advanced disease. The temporal pattern of integrin expression does not correspond with neointima area, but closely parallels vascular cell proliferation evaluated by Ki67 staining.
Because of the diffuse nature of GA, hearts with advanced disease are not amenable to traditional revascularization (PTCA, CABG); often retransplantation is the only therapeutic option. Early detection of GA will allow for better understanding of pathogenesis and potential therapeutic interventions. There is currently no noninvasive imaging method for early detection of GA. The temporal pattern of vß3-integrin expression in GA suggests that vß3 can be a suitable target for molecular imaging of the proliferative process in arteriopathy. RP748 uptake in transplanted human arteries (relative to the uptake in native vessels) is minimal in the absence of allogeneic stimulation, increases by 2 wk, and declines toward baseline in more advanced arteriopathy. Blocking experiments with excess nonlabeled parent molecule establish the specificity of the uptake. RP748 uptake closely parallels the proliferative process in transplanted arteries.
Integrin function is modulated by changes in avidity or affinity (integrin activation). We have previously demonstrated that RP748 and its cy3-labeled homologue, TA145 preferentially bind to the active conformation of vß3 integrin. Our findings here indicate the integrin expressed early on in GA is in the active conformation, and hence detected by RP748. Given the functional significance of vß3 integrin in cell proliferation and migration, this is not an unexpected finding. By targeting the active conformation of the integrin, RP748 provides an important tool for in vivo studies of integrin activation and adds an additional level of specificity for in vivo imaging by reducing binding to the nonactive conformer which is abundant on a number of cell types.
The data presented here support and expand the scope of our previous findings in mechanical injury-induced vascular remodeling. They suggest that changes in vß3 expression/activation are part of a common pathway shared by both processes. The chimeric human/mouse model, where both the effector and target cells are of human origin, provides a novel and unique opportunity to study tracer localization to remodeling human arteries in vivo. Although current imaging technologies’ limitations in sensitivity and resolution prohibit imaging of these very small arterial targets in vivo (data not shown), autoradiographic analysis has clearly established the in vivo localization of the tracer to target tissue. Ultimately, because human GA is a diffuse process involving the entire coronary tree, it should be more readily amenable to in vivo imaging. Future studies in larger animals will address the validity of this assumption.
In conclusion, we have demonstrated that vß3 integrin is a marker of cell proliferation in GA, and that RP748 uptake in vivo tracks the proliferative process. Although there is no optimal animal model of GA, by using the chimeric human/mouse model of GA, it is more likely that our findings will be applicable to human disease. vß3-targeted imaging may provide an opportunity to detect early GA in the clinical setting, advance our understanding of pathophysiology, and improve the care of cardiac transplant patients. Given the prevalence of vascular remodeling in a broad spectrum of arterial diseases, implications of our findings extend well beyond GA.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4130fje;
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