This Article Abstract Full Text (PDF) All Versions of this Article: 110/1/84    most recent 01.CIR.0000133319.84326.70v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sadeghi, M. M. Articles by Bender, J. R. Search for Related Content PubMed PubMed Citation Articles by Sadeghi, M. M. Articles by Bender, J. R. Related Collections Remodeling Restenosis Other Vascular biology Nuclear cardiology and PET Cell biology/structural biology Imaging

(Circulation. 2004;110:84-90.)
© 2004 American Heart Association, Inc.

Original Articles

Detection of Injury-Induced Vascular Remodeling by Targeting Activated _vß₃ Integrin In Vivo

Mehran M. Sadeghi, MD; Svetlana Krassilnikova, MD; Jiasheng Zhang, MD; Amir A. Gharaei, MD; Hooman Rastegar Fassaei, MD; Leila Esmailzadeh, MS; Ali Kooshkabadi; Scott Edwards, PhD; Padmaja Yalamanchili, PhD; Thomas D. Harris, PhD; Albert J. Sinusas, MD; Barry L. Zaret, MD; Jeffrey R. Bender, MD

From the Raymond and Beverly Sackler Cardiovascular Molecular Imaging Laboratory, Section of Cardiovascular Medicine (M.M.S., S.K., J.Z., A.A.G., H.R.F., L.E., A.K., A.J.S., B.L.Z., J.R.B.), Yale University School of Medicine, New Haven, Conn; VA Connecticut Healthcare System (M.M.S., S.K., J.Z., A.A.G., H.R.F., L.E., A.K.), West Haven, Conn; and Bristol-Myers Squibb Medical Imaging (S.E., P.Y., T.D.H.), North Billerica, Mass.

Correspondence to Mehran M. Sadeghi, MD, VA Connecticut Healthcare System, 950 Campbell Ave, 111B, West Haven, CT 06516. E-mail mehran.sadeghi{at}yale.edu

Received June 13, 2003; de novo received December 4, 2003; revision received February 23, 2004; accepted March 11, 2004.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— The _vß₃ integrin plays a critical role in cell proliferation and migration. We hypothesized that vascular cell proliferation, a hallmark of injury-induced remodeling, can be tracked by targeting _vß₃ integrin expression in vivo.

Methods and Results— RP748, a novel ¹¹¹In-labeled _vß₃-specific radiotracer, was evaluated for its cell-binding characteristics and ability to track injury-induced vascular proliferation in vivo. Three groups of experiments were performed. In cultured endothelial cells (ECs), TA145, a cy3-labeled homologue of RP748, localized to _vß₃ at focal contacts. Activation of _vß₃ by Mn²⁺ led to increased EC binding of TA145. Left common carotid artery wire injury in apolipoprotein E^–/– mice led to vascular wall expansion over a period of 4 weeks. RP748 (7.4 MBq) was injected into groups of 9 mice at 1, 3, or 4 weeks after left carotid injury, and carotids were harvested for autoradiography. Relative autographic intensity, defined as counts/pixel of the injured left carotid area divided by counts/pixel of the uninjured right carotid area, was higher at 1 and 3 weeks (1.8±0.1 and 1.9±0.2, respectively) and decreased significantly by 4 weeks after injury (1.4±0.1, P<0.05). Carotid _v and ß₃ integrin expression was maximal at 1 week and decreased by 4 weeks after injury. The proliferation index, as determined by Ki67 staining, followed a temporal pattern similar to that of RP748 uptake. Dynamic gamma imaging demonstrated rapid renal clearance of RP748.

Conclusions— RP748 has preferential binding to activated _vß₃ integrin and can track the injury-induced vascular proliferative process in vivo.

Key Words: imaging • nuclear medicine • arteries • restenosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Integrins are a family of heterodimeric adhesion molecules involved in cell–cell and cell–matrix interactions.^1,2 Through interactions with their specific counterreceptors, integrins play a central role in normal homeostasis and pathological states. Integrin function can be modulated by changes in the expression level and/or activation state of the integrin. These activation states are classically studied by use of activating antibodies and antibodies that recognize the activation epitopes of various integrins. The _vß₃ integrin is expressed on vascular endothelial cells (ECs)³ and smooth muscle cells (SMCs),⁴ as well as nonvascular cells such as osteoclasts.⁵ Classically described as the vitronectin receptor, _vß₃ integrin can also bind to other extracellular matrix proteins (eg, fibrinogen, fibronectin) as well as prothrombin. Modulation of _vß₃ expression by vascular cells plays a central role in vascular processes associated with cell proliferation and migration, such as angiogenesis⁶ and vascular remodeling.⁷ As such, _vß₃ integrin has emerged as a promising target for imaging tumor angiogenesis.^8,9 _vß₃ Antagonists have been shown to limit neointimal hyperplasia and lumen stenosis in experimental models of vascular injury.^10–12 Therefore, we hypothesized that injury-induced vascular remodeling/proliferative process could be imaged in vivo by targeting _vß₃ expression with a novel ¹¹¹In-labeled _vß₃ integrin–specific small molecule, RP748.

Here we demonstrate that (1) RP748 and its homologues bind preferentially to activated _vß₃ on ECs in vitro and exhibit favorable binding characteristics for in vivo imaging, (2) RP748 uptake can track/detect the proliferative process associated with carotid artery injury by targeting activated _vß₃ integrin expression in vivo in apolipoprotein E–negative (apoE^–/–) mice, and (3) pharmacokinetic characteristics are suitable for in vivo imaging.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
Reagents were from Sigma Chemical Co unless otherwise specified. TA138 and its cy3-labeled (TA145) and ¹¹¹In-labeled (RP748) homologues were provided by Bristol-Myers Squibb Medical Imaging. TA138 is a high-affinity, _vß₃ integrin–specific small molecule ligand, originally selected from a combinatorial library on the basis of its ability to inhibit human umbilical vein EC (HUVEC) adhesion to fibrinogen (_vß₃-dependent) with an IC₅₀ of 52±6 nmol/L.¹³

Cell Culture
Single-donor HUVECs were isolated and cultured as described previously.¹⁴ Murine lung ECs were kindly provided by Dr William Sessa (Yale University) and were grown in HUVEC medium.

Flow Cytometry
Cells were stained and analyzed on a FACSort (Becton Dickinson) as described previously.¹⁵ Cells were suspended in calcium/magnesium-free PBS. The primary mouse monoclonal antibodies used, anti-_vß₃ (LM609, Chemicon), or the irrelevant isotype-matched control antibody (MOPC-21), were followed by staining with FITC-conjugated goat anti-mouse IgG secondary antibody. TA145, a cy3-labeled TA138, was used directly. Inhibition assays were performed in the presence of 50-fold excess nonlabeled antibody. To study the effect of integrin activation on TA145 binding, cells were treated with Mn²⁺ or EDTA (1 mmol/L) for 10 minutes before staining. Five thousand cells were analyzed per sample.

Affinity Determination
HUVECs were exposed to serial dilutions of RP748 with or without Mn²⁺ for 30 minutes at 4°C. After 4 washes, the remaining activity was measured in a gamma counter (Packard Biosciences Co). Nonspecific binding was calculated with 100-fold excess unlabeled ligand, assuming a linear relation with the labeled ligand concentrations. Saturation binding was determined and Scatchard analysis performed with GraphPad PRISM software.

Animal Model
Left common carotid artery injury was induced in apoE^–/– mice as described previously¹⁶ with minor modifications. Six- to 8-week-old female apoE^–/– mice (Jackson Laboratory, Bar Harbor, Me) were fed high-cholesterol (1.25% cholesterol, Harlan) chow ad libitum. After 1 week, animals were anesthetized with ketamine (100 mg/kg IP) and xylazine (10 mg/kg IP). The common carotid and external carotid arteries were exposed by blunt-end dissection. A 0.014-in guide wire was introduced through the left external carotid artery and moved proximally into the left common carotid artery. The common carotid was abraded 6 times over its length. The opposite uninjured right carotid artery was used as control. Animals were reanesthetized at 1, 3, or 4 weeks after injury. For (immuno)histological analysis, groups of 3 animals at each time point were perfused with normal saline (or 3% paraformaldehyde for morphometric analysis) at 37°C for 5 minutes under physiological pressure through an intracardiac puncture. Carotid arteries were dissected, embedded in OCT compound, snap-frozen, and stored at –80°C until further use. For imaging experiments, groups of 9 animals at each time point were used. RP748 (7.4 MBq) was administered through an intravenous catheter. Blood samples were obtained at various time points after tracer administration and weighed, and activity was quantified in a gamma-well counter. Animals were killed after 2.5 hours, and carotids were harvested for autoradiography. Displacement experiments were performed in the presence of 50-fold excess nonlabeled TA138. Experiments were performed according to the regulations of Yale University’s Animal Care Committee.

Histology, Immunohistochemistry, and Immunofluorescence Studies
Hematoxylin and eosin, immunohistochemistry, and immunofluorescence staining were performed by use of standard techniques on 4-µm-thick, fixed cryostat sections. They were incubated overnight in the presence of nonbinding controls or anti-human and murine _v, ß₃ (both from Chemicon), or Ki67 (Santa Cruz Biotechnology) antibodies. For immunofluorescence, coverslips were exposed to TA145 or primary antibodies followed by fluorescence-labeled secondary antibodies. For morphometric analysis, microscopic measurements were performed on cryostat sections from paraformaldehyde-perfused animals at 250 µm below the carotid bifurcation using NIH Image software. Lumen diameter and (neo)intima and media thickness were measured on 2 perpendicular lines crossing the maximally thickened section of the vessel wall and were averaged for final analysis. Total and Ki67-positive nuclei in 4 random sections along the common carotid artery were counted, and the maximal ratio of Ki67 antibody–positive nuclei (in percents) was used as index of cell proliferation.

Autoradiography
Samples were exposed to x-radiographic X-OMAT Kodak Scientific Imaging Film (Eastman Kodak) for various times to optimize detection. Signal intensity in the regions of interest was measured on high-resolution images, and background noise was subtracted using 1D Image Analysis Software (Eastman Kodak Co). Relative autoradiographic intensity was defined as the average autoradiographic counts/pixel in the left common carotid divided by that of the uninjured right common carotid artery.

Serial Gamma Imaging for Pharmacokinetics
RP748 (7.4 MBq) was injected through an intravenous catheter. Serial 30-second planar high-resolution images were obtained using a 1-mm aperture pinhole collimator and a SPECT camera (GE Millenium VG) with a 256x256 matrix for 15 minutes, followed by a 30-minute late image at 2 hours. This system provides much higher resolution than can be obtained with the standard parallel-hole collimators. At 4-cm distance, using radioisotope-filled capillary tubes, the discrimination for the GE camera is found to be 1.5 mm for ¹¹¹In, with a count-sensitivity in the range of 1 to 2 counts per second/µCi. Images were acquired with a 10% window centered on photopeak of the radioisotope (174 and 242 keV for ¹¹¹In). Time-activity curves were derived for various regions of interest.

Statistical Analysis
All data are presented as mean±SEM. Multiple groups were compared by use of ANOVA with Tukey’s test for multiple post hoc comparisons. Significance was set at the 0.05 level.

	Results

Top Abstract Introduction Methods Results Discussion References

 
TA138 Interacts With _vß₃ Integrin on Human and Murine ECs
We first determined whether TA138 can detect _vß₃ integrin expressed on cells of human and murine origin. In HUVECs, LM609 (an anti-human _vß₃ integrin antibody) and TA145 colocalized to _vß₃ integrin in focal contacts (Figure 1, a, b, and c). The specificity of staining was demonstrated by lack of staining with an isotype-matched control antibody, MOPC-21 (data not shown). Similar results were obtained with murine ECs. In the absence of a specific anti-murine _vß₃ antibody, staining with anti-murine _v and ß₃ antibodies as well as TA145 localized to focal contacts (Figure 1, d, e, and f). Specificity of binding was further assessed by flow cytometry. LM609 but not MOPC-21 inhibited TA145 binding to HUVECs (Figure 2a), demonstrating the _vß₃-specificity of TA145 binding to live ECs.

View larger version (16K): [in this window] [in a new window]   Figure 1. Detection of integrin expression in human (a, b, c) and murine (d, e, f) ECs by immunofluorescent staining. Anti-vß3 antibody (a) and TA145 (b) colocalize (c) to focal contacts in human ECs. Similarly, in murine ECs, anti-v (d) and anti-ß3 (e) antibodies, as well as TA145 (f), localize to focal contacts.

View larger version (36K): [in this window] [in a new window]   Figure 2. Flow cytometric evaluation of TA145 binding to endothelial cells (ECs). A specific anti-vß3 antibody (LM609) but not control antibody (MOPC-21) inhibits TA145 binding (a). Presence of Mn2+ increases TA145 binding to ECs in a concentration-dependent manner (b) without affecting vß3 expression (c). Conversely, TA145 binding to ECs is inhibited by EDTA (d). Figure is representative of 4 independent experiments.

TA138 Recognition of _vß₃ Integrin Is Activation Dependent
The binding of monovalent reagents, such as TA138, is not affected by clustering of target molecules. Therefore, TA138 may be able to detect _vß₃ conformational changes independently of changes in integrin avidity. To test this hypothesis, we assessed the ability of TA145 to bind to _vß₃ integrin on Mn²⁺-treated HUVECs by use of flow cytometry. Presence of Mn²⁺, a universal activator of integrins through binding to metal ion–dependent adhesion sites and consequent conformational changes, increased the binding of TA145 to HUVECs in a concentration-dependent manner, with the maximal binding observed at 20 µmol/L (Figure 2b). This increased binding was not a result of increased surface expression of _vß₃ integrin because LM609 antibody binding was unaffected by the presence of Mn²⁺ (Figure 2c). Chelation of divalent ions by EDTA (1 mmol/L) reduced the baseline binding of TA145 (in the absence of Mn²⁺) to the level of unstained cells and resulted in a complete inhibition of the effect of Mn²⁺ (Figure 2d). Conversely, integrin activation by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA, 200 nmol/L), which exerts its effects primarily through changes in avidity rather than conformation, did not affect the TA145 binding to HUVECs (data not shown).

RP748 Binding Characteristics to Activated _vß₃ Integrin Are Favorable for In Vivo Imaging
The binding characteristics of RP748, the ¹¹¹In-labeled homologue of TA138, to HUVECs and the effect of integrin activation were assessed by saturation binding and Scatchard analysis (Figure 3). There was an approximately 15-fold increase in RP748 affinity for _vß₃ integrin on ECs in suspension in the presence of Mn²⁺ (2 mmol/L), compared with nonactivated cells, with the K_d decreasing from 374±44 nmol/L in the absence of Mn²⁺ to 21±7 nmol/L in the presence of Mn²⁺ (2 mmol/L). Integrin activation with Mn²⁺ also led to an increase in maximum binding (B_max) from 130 000±5000 molecules/EC at baseline to 230 000±16 000 molecules/EC in the presence of Mn²⁺.

View larger version (14K): [in this window] [in a new window]   Figure 3. RP748 saturation binding to endothelial cells. Various amounts of RP748 were added to HUVECs (in triplicates) in absence or presence of Mn2+, and specific binding was calculated. Bmax and Kd (230 000±16 000 binding sites/cell and 21±7 nmol/L, respectively, with Mn2+ and 130 000±5000 binding sites/cell and 374±44 nmol/L without Mn2+) were derived by Scatchard analysis. Figure is representative of 3 independent experiments.

Injury-Induced Vascular Remodeling Is Associated With Increased Expression of _v and ß₃ Integrins
Expression patterns of _v and ß₃ in the vessel wall were studied by immunohistochemistry at different times after carotid wire injury in apoE^–/– mice. Very low levels of _v and ß₃ staining could be detected in uninjured carotid arteries. On injury, and in conjunction with vascular wall expansion, higher levels of both the _v (Figure 4) and ß₃ (not shown) integrins could be detected in the media and neointima. Expression of _v and ß₃ integrin was maximal at 1 to 3 weeks after injury and decreased toward baseline levels by 4 weeks.

View larger version (122K): [in this window] [in a new window]   Figure 4. Immunohistochemical analysis of v integrin expression in control uninjured and injured carotid arteries at different times after injury. Maximal expression was observed at 1 to 3 weeks (w) after injury and decreased toward baseline by 4 weeks after injury. Control (R), shown at 4 weeks, was identical at all time points. Figure is representative of 3 independent experiments. R indicates right; L, left.

RP748 Uptake Is Increased After Carotid Wire Injury
To assess the ability of RP748 to track activated _vß₃ integrin expression in vivo, RP748 (7.4 MBq) was injected into apoE^–/– mice at 1, 3, and 4 weeks after left common carotid injury. Animals were killed at 2.5 hours, and the aortic arch and carotids were harvested for autoradiography. Despite some variability, there was significantly higher uptake of the tracer in the left injured common carotid artery compared with the contralateral uninjured artery (Figure 5a). Relative autoradiographic intensity was highest at 1 to 3 weeks and decreased significantly by 4 weeks after injury (1.8±0.1, 1.9±0.2, and 1.4±0.1 at 1, 3, and 4 weeks after injury, n=9 in each group, P=0.03) (Figure 5b). Specificity of uptake was demonstrated in displacement experiments, in which a 50-fold excess nonlabeled TA138 decreased RP748 localization to target arteries by 72% (Figure 5, c and d).

View larger version (33K): [in this window] [in a new window]   Figure 5. Autoradiographic analysis of RP748 uptake after left carotid injury. a, Examples of carotid autoradiographs at 1, 3, and 4 weeks (w) after left carotid injury. Arrows point to sites of injury. b, Range and quartiles of relative autographic intensity (left/right). Box extends from 25th to 75th percentile, with a line at median. Whiskers show highest and lowest values. n=9 at each time point. c, Representative example and d, densitometric analysis of RP748 uptake in absence (n=2) or presence (n=4) of 50-fold excess of nonlabeled TA138. Open triangle, left (L) carotid; square, right (R) Carotid. Scale bar, 5 mm.

RP748 Uptake Tracks Vascular Cell Proliferation
Left common carotid wire injury in apoE^–/– mice led to substantial vascular remodeling with neointima formation and expansion of media over a period of 4 weeks (Figure 6a). Media thickening could be observed as early as 1 week after injury (thickness: 7.9±0.4, 21.3±4.8, 63.4±5.6, and 79.1±26.5 µm, n=3, P<0.0001 for uninjured control right carotid and left carotid at 1, 3, and 4 weeks after injury, respectively) and was followed by the formation of a thick neointima by 3 weeks after injury (thickness: 0.0, 0.8±0.8, 62.7±8.3, and 132.4±12.1 mm, n=3, P<0.0001 for uninjured control right carotid and left carotid at 1, 3, and 4 weeks after injury, respectively) (Figure 6b). The proliferative process was quantified by Ki67 staining, a marker of cell proliferation. In uninjured arteries, Ki67 staining was minimal (proliferation index <1) at all time points. The left carotid proliferation index was maximal at 1 to 3 weeks and decreased significantly by 4 weeks after injury (18.6±0.3 and 21.4±2.4 versus 5.3±0.3, n=3, P<0.01, at 1, 3, and 4 weeks after injury, respectively). As such, the temporal pattern of RP748 relative carotid intensity closely paralleled that of the proliferation index (Figure 7).

View larger version (59K): [in this window] [in a new window]   Figure 6. Morphometric analysis. a, Representative hematoxylin and eosin staining of carotids 4 weeks after left carotid injury. b, Carotid intima and media thickness 250 µm below bifurcation in uninjured right carotid (similar at all time points) and 1, 3, and 4 weeks after left carotid injury. n=3.

View larger version (22K): [in this window] [in a new window]   Figure 7. Vascular cell proliferation analysis. Proliferation index (percent Ki67-positive nuclei, n=3) at 1, 3, and 4 weeks after left carotid injury closely tracks RP748 relative intensity (n=9). Inset: Representative Ki67 immunostaining.

RP748 Exhibits Favorable Pharmacokinetics for In Vivo Imaging
Dynamic high-resolution planar images demonstrated rapid renal clearance of RP748 in apoE^–/– mice. Figure 8a represents a typical time-activity curve. RP748 could be detected in the kidneys as early as 3 minutes after intravenous administration. By 2.5 hours, most of the activity was detected in the bladder. RP748 clearance was quantified by serial blood sampling. In concordance with imaging data, RP748 rapidly cleared from the circulation. Less than 0.5% of the injected dose was detectable in the blood pool at 2.5 hours (Figure 8b).

View larger version (20K): [in this window] [in a new window]   Figure 8. RP748 pharmacokinetics. a, Representative time-activity curve derived from dynamic gamma imaging of RP748 in apoE–/– mice, demonstrating rapid renal clearance of tracer. b, RP748 clearance quantified by serial blood sampling, confirming rapid clearance of tracer. ID indicates injected dose. n=3.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this series of experiments, we demonstrate the following: (1) RP748 binds to activated _vß₃ integrin with favorable characteristics for in vivo imaging, (2) RP748 uptake tracks the injury-induced carotid artery proliferative process in apoE^–/– mice, and (3) RP748 pharmacokinetics are suitable for in vivo imaging. These findings serve as a foundation for the development of noninvasive imaging modalities directed at vascular proliferation and remodeling.

The activation state(s) of integrins is (are) associated with changes in affinity (conformational changes) and in avidity (lateral mobility and clustering) of the molecule. Analysis of _vß₃ integrin crystal structure has led to description of 3 distinct molecular conformations.¹⁷ Mn²⁺ is the prototype activator of integrins. Other stimuli, such as phorbol esters and ADP,¹⁸ can similarly modulate the activation state of the integrins, although their effect on _vß₃ integrin conformation is less well studied. Recently, a synthetic antibody (WOW-1) was shown to specifically interact with activated _vß₃ integrin.¹⁹ Here, we demonstrated that both the cy3- and ¹¹¹In-labeled TA138 (ie, TA145 and RP748) bind preferentially to Mn²⁺-activated _vß₃ integrin on ECs. Saturation binding assays demonstrated a higher apparent affinity and number of binding sites per EC for RP748 in the presence of Mn²⁺. These binding parameters provide favorable conditions for in vivo imaging of activated _vß₃ integrin by RP748.²⁰ A similar number of _vß₃ binding sites per Mn²⁺-activated EC was observed with another activation-dependent ligand of _vß₃ integrin, prothrombin.¹⁸ Both the baseline and Mn²⁺-induced binding to ECs were inhibited by EDTA. Of note is that the focal contact–localized _vß₃ molecules in adherent ECs are apparently in the active conformation, as demonstrated by TA145 and LM609 costaining (Figure 1). Thus, a component of _vß₃ integrin on resting ECs is in the activated state, also seen in flow cytometric analysis (Figure 2) and therefore recognized by TA138 homologues. Interestingly, and contrary to WOW-1,¹⁹ we did not observe any increased binding of TA145 to PMA-treated ECs, suggesting that TA138 homologues may discriminate between the PMA- and Mn²⁺-activated forms of _vß₃ integrin. The effect of PMA on ß₁-dependent leukocyte adhesion is mainly due to integrin clustering (ie, changes in avidity), without significantly affecting the affinity of the receptor for the ligand.²¹ A similar pattern may prove to be true for _vß₃ integrin.

Carotid wire injury in apoE^–/– mice led to significant neointima formation and media thickening over a period of 4 weeks. There was concomitant upregulation of _v and ß₃ integrins in the remodeling vessel wall. Maximal expression of both integrins was observed at 1 week after injury and preceded maximal morphometric changes. In the absence of specific anti-murine _vß₃ integrin antibodies, evaluation of _v and ß₃ integrins constituted a reasonable substitute. Although we did not specifically study the cellular localization of the observed _vß₃ integrin expression, the predominance of immunostaining in the media and neointima strongly implicates both SMCs and ECs.

We observed a higher uptake of RP748 in the injured left carotid artery compared with the uninjured contralateral artery, with a time course following that of the proliferative process. Specificity of uptake was demonstrated by the inhibitory effect of excess nonlabeled tracer. Similar to the _vß₃ expression pattern, the maximal proliferative process preceded the maximal morphometric changes. Interestingly, RP748 uptake seems to track the proliferative process more closely than the immunohistochemical evaluation of _v and ß₃ integrin expression. Immunohistochemical detection of _vß₃ integrin by anti-_v and -ß₃ antibodies does not necessarily correlate with expression of the activated form of the integrin. The superior performance of RP748 may be due to its preferential binding to the activated form of _vß₃ integrin. There is limited information available on the physiopathological importance of _vß₃ activation in vivo. TA138 and its labeled homologues may provide a unique opportunity for studying these processes, both in vitro and in vivo. The rapid renal clearance of RP748, limited liver uptake, and its high affinity for activated _vß₃ integrin favorably affect its suitability for in vivo imaging in a number of biological processes. As such, high levels of RP748 uptake have been observed in _vß₃-expressing tumors.¹³ The dose of RP748 used in this study was selected on the basis of preliminary imaging experiments to allow for reasonable analysis of tracer biodistribution/kinetics by in vivo gamma imaging. The optimal dose and imaging parameters will need to be further defined. Future experiments will be directed at imaging injury-induced vascular remodeling in both normal and atherosclerotic arteries in larger animals.

In conclusion, we have demonstrated that RP748 exhibits selective binding to activated _vß₃ integrin and can track the injury-induced arterial proliferative process. These findings will potentially lead to the development of noninvasive imaging strategies for vascular cell proliferation–associated states, whether focal, as in postangioplasty restenosis, or diffuse, as in pulmonary hypertension. As such, the ability to track/predict the proliferative process in vivo can markedly affect the management of patients with graft vasculopathy, in which noninvasive imaging techniques are lacking. Moreover, RP748 provides investigators with a new experimental tool to study _vß₃ integrin activation in various biological processes in vivo. This and other molecular imaging–based approaches should lead to better understanding of pathophysiology and development of novel paradigms for management of cardiovascular disease.

	Acknowledgments

 
This work was supported by a grant from the Raymond and Beverly Sackler Foundation and National Institutes of Health grant NIH-P01-HL-70295. Dr Sadeghi is the recipient of an American College of Cardiology Harry B. Graf Career Development Award for Heart Disease Prevention.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999; 285: 1028–1032.[Abstract/Free Full Text]
   
2. Humphries MJ. Integrin structure. Biochem Soc Trans. 2000; 28: 311–339.[Medline] [Order article via Infotrieve]
   
3. Cheresh DA. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc Natl Acad Sci U S A. 1987; 84: 6471–6475.[Abstract/Free Full Text]
   
4. Moiseeva EP. Adhesion receptors of vascular smooth muscle cells and their functions. Cardiovasc Res. 2001; 52: 372–386.[Abstract/Free Full Text]
   
5. Horton MA, Taylor ML, Arnett TR, et al. Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts. Exp Cell Res. 1991; 195: 368–375.[CrossRef][Medline] [Order article via Infotrieve]
   
6. Eliceiri BP, Cheresh DA. Adhesion events in angiogenesis. Curr Opin Cell Biol. 2001; 13: 563–568.[CrossRef][Medline] [Order article via Infotrieve]
   
7. Sajid M, Stouffer GA. The role of alpha(v)beta3 integrins in vascular healing. Thromb Haemost. 2002; 87: 187–193.[Medline] [Order article via Infotrieve]
   
8. Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat Med. 1998; 4: 623–626.[CrossRef][Medline] [Order article via Infotrieve]
   
9. Haubner R, Wester HJ, Reuning U, et al. Radiolabeled alpha(v)beta3 integrin antagonists: a new class of tracers for tumor targeting. J Nucl Med. 1999; 40: 1061–1071.[Abstract/Free Full Text]
   
10. Matsuno H, Stassen JM, Vermylen J, et al. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation. 1994; 90: 2203–2206.[Abstract/Free Full Text]
    
11. Choi ET, Engel L, Callow AD, et al. Inhibition of neointimal hyperplasia by blocking alphaVbeta3 integrin with a small peptide antagonist GpenGRGDSPCA. J Vasc Surg. 1994; 19: 125–134.[Medline] [Order article via Infotrieve]
    
12. Srivatsa SS, Fitzpatrick LA, Tsao PW, et al. Selective alphavbeta3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin alphavbeta3 and osteopontin expression during neointima formation. Cardiovasc Res. 1997; 36: 408–428.[Abstract/Free Full Text]
    
13. Harris TD, Kalogeropoulos S, Nguyen T, et al. Design, synthesis, and evaluation of radiolabeled integrin alpha v beta 3 receptor antagonists for tumor imaging and radiotherapy. Cancer Biother Radiopharm. 2003; 18: 627–641.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Gimbrone MA Jr, Cotran RS, Folkman J. Human vascular endothelial cells in culture: growth and DNA synthesis. J Cell Biol. 1974; 60: 673–684.[Abstract/Free Full Text]
    
15. Sadeghi MM, Collinge M, Pardi R, et al. Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein. J Immunol. 2000; 165: 2712–2718.[Abstract/Free Full Text]
    
16. De Geest B, Zhao Z, Collen D, et al. Effects of adenovirus-mediated human apo A-I gene transfer on neointima formation after endothelial denudation in apo E-deficient mice. Circulation. 1997; 96: 4349–4356.[Abstract/Free Full Text]
    
17. Takagi J, Petre BM, Walz T, et al. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002; 110: 599–511.[CrossRef][Medline] [Order article via Infotrieve]
    
18. Byzova TV, Plow EF. Activation of alphaVbeta3 on vascular cells controls recognition of prothrombin. J Cell Biol. 1998; 143: 2081–2092.[Abstract/Free Full Text]
    
19. Pampori N, Hato T, Stupack DG, et al. Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand. J Biol Chem. 1999; 274: 21609–21616.[Abstract/Free Full Text]
    
20. Blankenberg FG, Strauss HW. Nuclear medicine applications in molecular imaging. J Magn Reson Imaging. 2002; 16: 352–361.[CrossRef][Medline] [Order article via Infotrieve]
    
21. Liu L, Schwartz BR, Tupper J, et al. The GTPase Rap1 regulates phorbol 12-myristate 13-acetate-stimulated but not ligand-induced beta 1 integrin-dependent leukocyte adhesion. J Biol Chem. 2002; 277: 40893–40900.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				P. M. Winter, A. H. Schmieder, S. D. Caruthers, J. L. Keene, H. Zhang, S. A. Wickline, and G. M. Lanza Minute dosages of {alpha}{nu}{beta}3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits FASEB J, August 1, 2008; 22(8): 2758 - 2767. [Abstract] [Full Text] [PDF]

				L. L. Johnson, L. Schofield, T. Donahay, M. Bouchard, A. Poppas, and R. Haubner Radiolabeled Arginine-Glycine-Aspartic Acid Peptides to Image Angiogenesis in Swine Model of Hibernating Myocardium J. Am. Coll. Cardiol. Img., July 1, 2008; 1(4): 500 - 510. [Abstract] [Full Text] [PDF]

				L. Kalinowski, L. W. Dobrucki, D. F. Meoli, D. P. Dione, M. M. Sadeghi, J. A. Madri, and A. J. Sinusas Targeted imaging of hypoxia-induced integrin activation in myocardium early after infarction J Appl Physiol, May 1, 2008; 104(5): 1504 - 1512. [Abstract] [Full Text] [PDF]

				T. Cyrus, H. Zhang, J. S. Allen, T. A. Williams, G. Hu, S. D. Caruthers, S. A. Wickline, and G. M. Lanza Intramural Delivery of Rapamycin With {alpha}v{beta}3-Targeted Paramagnetic Nanoparticles Inhibits Stenosis After Balloon Injury Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 820 - 826. [Abstract] [Full Text] [PDF]

				M. P. S. Dunphy, H. Schoder, and H. W. Strauss Radionuclide Techniques for Identifying Vulnerable Plaque J. Nucl. Med., November 1, 2007; 48(11): 1753 - 1755. [Full Text] [PDF]

				J. C. Wu, F. M. Bengel, and S. S. Gambhir Cardiovascular Molecular Imaging Radiology, August 1, 2007; 244(2): 337 - 355. [Abstract] [Full Text] [PDF]

				M. C. Laterza, L. D.N.J. de Matos, I. C. Trombetta, A. M.W. Braga, F. Roveda, M. J.N.N. Alves, E. M. Krieger, C. E. Negrao, and M. U.P.B. Rondon Exercise Training Restores Baroreflex Sensitivity in Never-Treated Hypertensive Patients Hypertension, June 1, 2007; 49(6): 1298 - 1306. [Abstract] [Full Text] [PDF]

				W. Cai, J. Rao, S. S. Gambhir, and X. Chen How molecular imaging is speeding up antiangiogenic drug development. Mol. Cancer Ther., November 1, 2006; 5(11): 2624 - 2633. [Abstract] [Full Text] [PDF]

				P. M. Winter, A. M. Neubauer, S. D. Caruthers, T. D. Harris, J. D. Robertson, T. A. Williams, A. H. Schmieder, G. Hu, J. S. Allen, E. K. Lacy, et al. Endothelial {alpha}{nu}{beta}3 Integrin-Targeted Fumagillin Nanoparticles Inhibit Angiogenesis in Atherosclerosis Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2103 - 2109. [Abstract] [Full Text] [PDF]

				G.A. Stouffer, A. Pathak, R. Zhao, and J. Huang Down But Not Out: New Insights Into the Role of {alpha}V{beta}3 Integrins in Vascular Healing Arterioscler. Thromb. Vasc. Biol., July 1, 2005; 25(7): 1309 - 1310. [Full Text] [PDF]

				J. E. Barbato, B. S. Zuckerbraun, M. Overhaus, K. G. Raman, and E. Tzeng Nitric oxide modulates vascular inflammation and intimal hyperplasia in insulin resistance and the metabolic syndrome Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H228 - H236. [Abstract] [Full Text] [PDF]

				J. Hua, L. W. Dobrucki, M. M. Sadeghi, J. Zhang, B. N. Bourke, P. Cavaliere, J. Song, C. Chow, N. Jahanshad, N. van Royen, et al. Noninvasive Imaging of Angiogenesis With a 99mTc-Labeled Peptide Targeted at {alpha}v{beta}3 Integrin After Murine Hindlimb Ischemia Circulation, June 21, 2005; 111(24): 3255 - 3260. [Abstract] [Full Text] [PDF]

				K.-H. Lee, K.-H. Jung, S.-H. Song, D. H. Kim, B. C. Lee, H. J. Sung, Y.-M. Han, Y. S. Choe, D. Y. Chi, and B.-T. Kim Radiolabeled RGD Uptake and {alpha}v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs J. Nucl. Med., March 1, 2005; 46(3): 472 - 478. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 110/1/84    most recent 01.CIR.0000133319.84326.70v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sadeghi, M. M. Articles by Bender, J. R. Search for Related Content PubMed PubMed Citation Articles by Sadeghi, M. M. Articles by Bender, J. R. Related Collections Remodeling Restenosis Other Vascular biology Nuclear cardiology and PET Cell biology/structural biology Imaging