Imaging of Integrin α v β 3 Expression Using 68 Ga-RGD Positron Emission Tomography in Pediatric Cerebral Infarct

Abstract

Enhanced expression of integrin α_vβ₃ is commonly used as a biomarker for angiogenesis, which is one of the key pathophysiologic processes in cerebral infarct. Integrin α_vβ₃ can be imaged with arginine-glycine-aspartic acid (RGD) peptide agents. In this study, characteristics of positron emission tomography (PET) using a ⁶⁸Ga-labeled RGD were investigated in pediatric cerebral infarct. Pediatric patients with moyamoya disease underwent ⁶⁸Ga-RGD PET in a research protocol for neovascularization evaluation. In these patients, 17 cerebral infarct lesions of 10 patients were included in the analysis. On ⁶⁸Ga-RGD PET, the infarct lesion to contralateral brain ratio (LCR) of the infarct lesion was measured and analyzed with regard to postinfarct time interval (PTI) and perfusion single-photon emission computed tomography (SPECT) findings. An increase in ⁶⁸Ga-RGD uptake was observed in cerebral infarct, particularly in recent lesions. The LCR was significantly higher in the recent than in the chronic lesions, and a significant correlation existed between the LCR and PTI. Additionally, the LCR was significantly higher in the lesions with hyperperfusion on SPECT. This study, as the first human study using an RGD agent for in vivo cerebral infarct imaging, demonstrated that ⁶⁸Ga-RGD PET has a potential for molecular imaging of integrin α_vβ₃ expression in cerebral infarct as a biomarker of angiogenesis.

IN ISCHEMIC CEREBRAL DISORDERS, angiogenesis is one of the key pathophysiologic processes for spontaneous recovery or therapeutic interventions.^1,2 With recent progress in molecular imaging, it has been actively studied to assess angiogenesis directly and noninvasively by imaging methods. Molecular targets such as vascular endothelial growth factor (VEGF) and integrin α_vβ₃ have been focused as targets in the development of imaging probes.^3,4 Integrin α_vβ₃ expression is enhanced on vascular endothelium with angiogenic activation and can be imaged with a tripeptide moiety of arginine-glycine-aspartic acid (RGD).^5,6

We also developed an RGD agent labeled with ⁶⁸Ga (⁶⁸Ga-RGD) and successfully obtained positron emission tomography (PET) images for angiogenesis in a tumor model.⁷ This agent was a conjugate of a cyclic RGD and 2-(p-isothiocyanoatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) labeled with ⁶⁸Ga. ⁶⁸Ga-RGD was blocked by a cold RGD agent in an in vitro binding assay and an animal model, suggesting specific binding to the integrin α_vβ₃.⁷

In ischemic stroke, angiogenesis imaging with ⁶⁴Cu-DOTA-VEGF PET was tried in a rat model, visualizing enhanced angiogenesis after stroke.⁸ RGD peptide imaging was successfully obtained in myocardial infarct and peripheral muscular ischemia in animal and human studies.^9,10 However, there has been no report on RGD peptide imaging in cerebral infarct. Moyamoya disease (MMD), a spontaneous and progressive steno-occlusive change in the internal carotid arteries and their branches, is one of the most important causes of cerebral infarct in children.¹¹ In the current study, we acquired ⁶⁸Ga-RGD PET in pediatric MMD patients and investigated the characteristics of ⁶⁸Ga-RGD PET images in cerebral infarct. To our knowledge, this is the first human study that investigated angiogenesis imaging using RGD agents in cerebral infarct.

Methods

A prospective study, in which cerebral angiogenesis after revascularization surgery was assessed using ⁶⁸Ga-RGD PET, enrolled 26 pediatric patients with MMD. The study was approved by the Institutional Review Board of the Seoul National University Hospital. Among them, 10 patients (age 1–14 years, M:F = 6:4) presented with 17 lesions of cerebral infarct before PET scans and were included in the current study. In the study protocol, ⁶⁸Ga-RGD was prepared as previously reported,⁷ and brain PET images were acquired 20 minutes after ⁶⁸Ga-RGD injection (111 MBq). Brain magnetic resonance imaging (MRI) and ^99mTc-HMPAO perfusion single-photon emission computed tomography (SPECT) were also performed before PET scans. PET images were visually analyzed by two nuclear medicine physicians blinded to clinical information, and ⁶⁸Ga-RGD uptake was quantified for the volume of interest (VOI) drawn for each infarct lesion with the aid of slice-matched MRI. The uptake was expressed as a lesion to control ratio (LCR) by quantifying another VOI that was drawn for the cerebral parenchyme of non-infarcted contralateral hemisphere.

The LCR of infarct lesions was analyzed with regard to postinfarct time interval (PTI) between infarct onset and PET scanning to assess characteristics of angiogenesis imaging in cerebral infarct. Infarct onset was determined by the first presentation of clinical symptoms or first MRI-based confirmation of acute infarct when the onset of clinical symptom was vague. Infarct lesions for which the PTI was < 30 days were classified as recent infarct, and the others were classified as chronic infarct. Additionally, the LCR was analyzed with regard to perfusion state shown on SPECT images. In statistical analyses, the Student t-test and Mann-Whitney test were performed to compare groups. Proportions were compared using the Fisher exact test. Additionally, Pearson correlation coefficient was calculated for correlation analysis.

Results

The clinical characteristics of the patients included in the current study are summarized in Table 1. Of the 17 infarct lesions, 13 lesions were diagnosed with cerebral infarct by presentation of neurologic symptoms and following MRI; the other 4 lesions were diagnosed by MRI for evaluation of underlying MMD without explicit neurologic symptoms. Based on the infarct onset time, 10 lesions were classified as recent infarcts and the other 7 as chronic ones at the time of ⁶⁸Ga-RGD PET.

Table 1.

Clinical Characteristics and Summary of Image Findings

					⁶⁸Ga-RGD PET		Perfusion SPECT
Patient No.	Sex	Age (yr)	Lesion No.	Infarct Site	PTI (d)	LCR	PTI (d)	Findings
1	F	2	1	L occipital	21	2.6	20	Perfusion decrease
			2	L frontal	1*	15.0	0	Hyperperfusion
2	M	2	3	L parietal	10	9.6	3	Not identifiable
			4	R parietal	29	1.0	22	Not identifiable
			5	L frontal lobe	56	1.4	49	Perfusion decrease
			6	R centrum seimovale	46	0.7	39	Perfusion decrease
3	F	4	7	R frontotemporal	14	9.7	5	Hyperperfusion
4	M	3	8	R frontoparietal	10	5.1	9	Hyperperfusion
			9	R medial frontal	10*	2.0	9	Perfusion decrease
			10	L frontotemporal	25	1.1	24	Perfusion decrease
			11	R temporoparietal	6	3.2	5	Perfusion decrease
5	M	4	12	L frontoparietal	24	1.7	24	Perfusion decrease
6	F	13	13	R frontoparietotemporal	422*	1.4	378	Perfusion decrease
7	F	6	14^†	L frontoparietal	142	0.8	143	Perfusion decrease
8	M	6	15^†	R frontal	144	1.6	138	Perfusion decrease
9	M	7	16	L frontal	113*	1.6	114	Perfusion decrease
10	F	14	17^†	R parietal	116	0.8	116	Perfusion decrease

LCR = lesion to control ratio; PET = positron emission tomography; PTI = postinfarct time interval; SPECT = single-photon emission computed tomography.

PTI determined by magnetic resonance imaging.

†

Postoperative infarct after revascularization surgery.

⁶⁸Ga-RGD PET after Cerebral Infarct

On PET scans, eight (47%) infarct lesions were definitely discernible on visual assessment with increased ⁶⁸Ga-RGD uptake. Notably, uptake of ⁶⁸Ga-RGD in the infarct lesions was significantly different according to the PTI of lesions. In a recent infarct group, eight (80%) were visually discernible, whereas none were discernible in the chronic infarct group (p = .002). In quantitative analysis, the LCR was also significantly higher in the recent infarct group than in the chronic group (5.10 ± 4.75 and 1.19 ± 0.40, respectively; p < .05). A significant correlation existed between PTI and LCR in the recent infarct group (r = −0.70; p < .05, Figure 1A). A representative case in which recent and chronic infarct lesions existed together showed marked ⁶⁸Ga-RGD uptake only in a recent infarct lesion (Figure 1, B and C).

Figure 1.

Correlation between postinfarct time interval (PTI) and angiogenic activation. A significant correlation existed between PTI and the lesion to control ratio (LCR) in the recent infarct group (A). On MRI (B) and ⁶⁸Ga-RGD PET (C) of a patient who had recent and chronic infarct lesions together, only the recent infarct (arrowhead, PTI = 10 days, lesion number 3) showed definite ⁶⁸Ga-RGD uptake, whereas the chronic infarct did not (arrow, PTI = 56 days, lesion number 5). High uptake in the cranium was observed in craniotomy sites for revascularization surgery (C).

⁶⁸Ga-RGD PET and Perfusion State on SPECT

For 10 recent infarct lesions, perfusion SPECT was performed 2.9 ± 3.2 (range 1–9) days before ⁶⁸Ga-RGD PET. Among them, three lesions showed postinfarct hyperperfusion and the other seven lesions showed a perfusion decrease of variable degree (see Table 1). The LCRs of the three lesions with hyperperfusion were significantly higher than those of the other lesions (9.93 ± 4.95 and 3.03 ± 3.00, respectively; p = .033). A representative case in which recent infarct lesions with or without hyperperfusion existed together showed more definite ⁶⁸Ga-RGD uptake in a lesion with hyperperfusion (Figure 2).

Figure 2.

Two recent infarct lesions of a patient showing different ⁶⁸Ga-RGD uptake according to postinfarct hyperperfusion. ⁶⁸Ga-RGD PET (A) and SPECT (B) showed marked uptake in a lesion with hyperperfusion (lesion to control ratio [LCR] = 5.1, arrow, lesion number 8). As a contrast, mild uptake was observed in a lesion with perfusion decrease (C and D, arrowhead, LCR = 3.2, lesion number 11). High uptake in the cranium was observed in craniotomy sites for revascularization surgery (C).

Discussion

In this study, ⁶⁸Ga-RGD PET, targeting integrin α_vβ₃ expression, was investigated in pediatric cerebral infarct. Increased uptake of ⁶⁸Ga-RGD was observed in recent infarct lesions with a significant correlation with postinfarct hyperperfusion, suggesting angiogenic activation in the acute postinfarct state.

After cerebral infarct, angiogenesis is known to be enhanced in the acute phase. On ⁶⁴Cu-DOTA-VEGF PET, uptake remained elevated until 16 days after infarct.⁸ Integrin α_vβ₃ expression was also found upregulated in angiogenic endothelium after cerebral infarct, with a decrease after 7 days.^12,13 Uptake of ⁶⁸Ga-RGD in the current study was consistent with these previous studies, demonstrating a peak in the acute stage and a decrease with time. Thus, ⁶⁸Ga-RGD PET is speculated to successfully visualize serial change of integrin α_vβ₃ expression in cerebral infarct. As integrin α_vβ₃ is expressed on activated macrophages¹⁴ as well as angiogenic endothelium, the increased uptake would be a mixed result of inflammation and angiogenesis. However, uptake of ⁶⁸Ga-RGD had a significant correlation with postinfarct hyperperfusion in the current study. Post-ischemic hyperperfusion on ^99mTc-HMPAO SPECT is induced by an increase in cerebral blood volume and blood flow,¹⁵ as well as new vessel formation.¹⁶ Thus, increased uptake of ⁶⁸Ga-RGD is indicative of endothelial angiogenic activation accompanying postinfarct hyperperfusion, at least as part of the mechanism.

Angiogenesis in cerebral infarct is a biomarker for disease progress or functional prognosis, as well as a therapeutic target.^17,18 Serum VEGF, which is related to angiogenesis, had a relationship with severity of stroke and was regarded as a prognostic factor predicting functional outcome.^19,20 Angiogenesis imaging can provide a better understanding of angiogenic activation after infarct and can be applied to angiogenesis-targeting treatment for patient selection or efficacy monitoring. ⁶⁸Ga-RGD PET demonstrated the potential for angiogenesis imaging for such purposes.

In this study, only pediatric patients with MMD were selected, which may influence the imaging findings. Additionally, disruption of the blood-brain barrier (BBB) would have influenced the uptake of ⁶⁸Ga-RGD like many other tracer imaging methods. BBB disruption is also one of the mechanisms for postinfarct hyperperfusion. To specifically assess angiogenic activity using ⁶⁸Ga-RGD PET in infarct lesions, the influence of BBB disruption should be adjusted using other methods, such as BBB imaging. The influence of hyperperfusion also needs to be considered because hyperperfusion per se may induce increased uptake of imaging tracers. Further studies are warranted to investigate the clinical roles of ⁶⁸Ga-RGD PET in general infarct patients and specific estimation methods for angiogenic activity with adjustment of BBB disruption and hyperperfusion effect.

This study demonstrates the feasibility and characteristics of ⁶⁸Ga-RGD PET in human cerebral infarct as a molecular imaging method for integrin α_vβ₃ expression. ⁶⁸Ga-RGD PET would be effective for both diagnosis and treatment monitoring in ischemic stroke, particularly with novel therapies targeting angiogenesis or endothelial activation.

Footnotes

Acknowledgment

Financial disclosure of authors: This study was supported by a National Research Foundation of Korea grant funded by the Korean Government (2009-007-6743).

References

Arai

Jin

Navaratna

. Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke. FEBS J 2009;276:4644–52, doi:10.1111/j.1742-4658.2009.07176.x.

Beck

Plate

. Angiogenesis after cerebral ischemia. Acta Neuropathol 2009;117:481–96, doi:10.1007/s00401-009-0483-6.

Sinusas

. Imaging of angiogenesis. J Nucl Cardiol 2004;11:617–33, doi:10.1016/j.nuclcard.2004.07.001.

Cai

Chen

Mohamedali

. PET of vascular endothelial growth factor receptor expression. J Nucl Med 2006;47:2048–56.

Haubner

Wester

. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. Curr Pharm Des 2004;10:1439–55, doi:10.2174/1381612043384745.

Haubner

Beer

Wang

. Positron emission tomography tracers for imaging angiogenesis. Eur J Nucl Med Mol Imaging 37 Suppl 1:S86–103.

Jeong

Hong

Chang

. Preparation of a promising angiogenesis pet imaging agent: ⁶⁸Ga-labeled c(rgdyk)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid and feasibility studies in mice. J Nucl Med 2008;49:830–6, doi:10.2967/jnumed.107.047423.

Cai

Guzman

Hsu

. Positron emission tomography imaging of poststroke angiogenesis. Stroke 2009;40:270–7, doi:10.1161/STROKEAHA.108.517474.

Verjans

Wolters

Laufer

. Early molecular imaging of interstitial changes in patients after myocardial infarction: comparison with delayed contrast-enhanced magnetic resonance imaging. J Nucl Cardiol 2010;17:1065–72, doi:10.1007/s12350-010-9268-5.

10.

Lee

Jung

Song

. Radiolabeled RGD uptake and alphav integrin expression is enhanced in ischemic murine hindlimbs. J Nucl Med 2005;46:472–8.

11.

Kuroda

Houkin

. Moyamoya disease: current concepts and future perspectives. Lancet Neurol 2008;7:1056–66, doi:10.1016/S1474-4422(08)70240-0.

12.

Welser

Milner

. Absence of the alpha v beta 3 integrin dictates the time-course of angiogenesis in the hypoxic central with compensatory increases in alpha 5 beta 1 integrin expression. J Cereb Blood Flow Metab 2010;30:1031–43, doi:10.1038/jcbfm.2009.276.

13.

Liu

Welser-Alves

. Upregulation of fibronectin and the alpha5beta1 and alphavbeta3 integrins on blood vessels within the cerebral ischemic penumbra. Exp Neurol 2012;233:283–91, doi:10.1016/j.expneurol.2011.10.017.

14.

Waldeck

Hager

Holtke

. Fluorescence reflectance imaging of macrophage-rich atherosclerotic plaques using an alphavbeta3 integrin-targeted fluorochrome. J Nucl Med 2008;49:1845–51, doi:10.2967/jnumed.108.052514.

15.

Cho

Hayashida

Imakita

. Hemodynamic and metabolic state of hyperfixation with ^99mTc-HMPAO brain spect in subacute stroke. Ann Nucl Med 2000;14:159–63, doi:10.1007/BF02987854.

16.

Lin

Sun

Cheung

. Dynamic changes in cerebral blood flow and angiogenesis after transient focal cerebral ischemia in rats. Evaluation with serial magnetic resonance imaging. Stroke 2002;33:2985–91, doi:10.1161/01.STR.0000037675.97888.9D.

17.

Arenillas

Sobrino

Castillo

. The role of angiogenesis in damage and recovery from ischemic stroke. Curr Treat Options Cardiovasc Med 2007;9:205–12, doi:10.1007/s11936-007-0014-5.

18.

Krupinski

Kaluza

Kumar

. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 1994;25:1794–8, doi:10.1161/01.STR.25.9.1794.

19.

Slevin

Krupinski

Slowik

. Serial measurement of vascular endothelial growth factor and transforming growth factor-beta1 in serum of patients with acute ischemic stroke. Stroke 2000;31:1863–70, doi:10.1161/01.STR.31.8.1863.

20.

Hermann

Zechariah

. Implications of vascular endothelial growth factor for postischemic neurovascular remodeling. J Cereb Blood Flow Metab 2009;29:1620–43, doi:10.1038/jcbfm.2009.100.