Preliminary Clinical Translation Of α_vβ₃-integrin Imaging In Evaluation Of Post-stroke Angiogenesis

Background and ObjectiveCerebrovascular disease, with high occurrence rate, morbidity and mortality, has been one of the leading causes of death and disability worldwide. Ischemic stroke accounts for most of the cerebrovascular disease. However, the really effective treatment is limited and has a narrow treatment time window (less than6hours). As a result, only a few patients can benefit from these treatments. It’s in urgent need to expand the limited treatment strategies. Therapeutic angiogenesis holds a promise to re-establish blood supply and revise brain function. However, there has not been an objective endpoint to quantitatively evaluate angiogenesis in vivo. Through our efforts and the others’, there may be a possible breakthrough in clinical translation of angiogenesis imaging targeting the αvβ3-integrin receptor.Methods:The human adipose tissues were collected from healthy adult women who underwent liposuction with informed consent. At the fifth passage of culture, the human adipose-derived mesenchymal stem cells (hADMSCs) were collected and analyzed by flow cytometry. Two hours after the middle cerebral artery occlusion (MCAO), the thread was removed to re-perfuse the middle cerebral artery. Screen the rats with an appropriate infarction size shown in the99mTc-ECD images which were performed on the first day after the establishment of MCAO model. Thirty-six rats were divided into3groups randomly:(1) normal saline-injection control group (n=15):three days after the MCAO models were established,10μL normal saline (NS) were injected into the peri-infarction areas through stereotactic method;(2) hADMSCs-treated group (n=15):three days after the MCAO models were established,10μL hADMSCs (3×106) were injected into the peri-infarction areas through stereotactic method;(3) VEGF-treated group (n=6):three days after the models were established, osmotic micropump (alzet1007D/kit2,100μL/lw) filled with4ug VEGF were implanted besides the infraction area at the same injection site. After the establishment of the models, the following tests were performed:(1) Modified neurological severity scores (mNSS) test and the adhesive-removal somatosensory test were performed to evaluate the recovery of neurological function on the1st before the modeling and the1st,3rd,7th,14th,21th and28th day after the modeling;(2)99mTc-ECD SPECT/CT brain blood perfusion imaging were performed on the6th、13th and27th day after the models establishment for the control and hADMSCs groups; while the VEGF group performed the study on the13th and27th day only;(3)99mTc-3PRGD2SPECT/CT angiogenesis imaging were performed one day after the99mTc-ECD imaging in these three groups, that is, on the7th、14th and28th day for the control and hADMSCs groups and the14th and28th day for the VEGF group, respectively;(4)18F-FDG PET imaging was performed on the14th and28th day post-stoke in all three groups.Results:1. From the14th day after the models were established, hADMSCs-and VEGF-treated rats exhibited more significant behavioral recovery than the control group rats with less mNSS scores and less object-removal time (P<0.05). Besides, the VEGF-treated rats recovered better than the hADMSCs-treated rats (P<0.05).2. Significant18F-FDG uptake defect were observed at the cortex and corpus striatum which were supplied by the middle cerebral artery in these three group rats on the14th day after the modeling. Improved18F-FDG uptake in the infarction area was detected on the28th-day imaging at different degree in all three groups with more18F-FDG uptake and smaller infarction size. hADMSCs-treated rats (0.43±0.03) showed more improvements than the control rats (0.54±0.089) but less than the VEGF-treated rats (0.31±0.053) with the P value of0.000and0.001, respectively.3. On the sixth day after the modeling, hADMSCs-treated rats and the control rats had no significant difference in the perfusion imaging (P>0.05). However, as the time passed on, more99mTc-ECD uptake and less defect area were observed in these three group of rats. Since the13th day, significant difference had been observed among these three groups. VEGF-treated rats showed the best blood perfusion improvments (P<0.05).4. For the hADMSCs-treated and control group rats, the most99mTc-3PRGD2uptake were observed on the7th day after the modeling (RRGD:(9.16±0.35);(5.2±0.42)) and showed significant difference between the different treatment groups (P=0.012). Less99mTc-3PRGD2accumulation was found on the14th day than that on the7th day. No significant difference was observed between these two groups on the28th day (P>0.05).5. On the14th and28th day after the modeling, the VEGF-treated rats showed significant higher99mTc-3PRGD2uptake than the hADMSCs-treated rats with a P value of0.002and0.086, respectively.6. Significant difference was observed between the DRGD on the7th day and DECD on the27th day and RFDG on the28th day (P=0.008) and0.012, respectively)Conclusion:RGD-based integrin imaging is of great use for angiogenesis imaging in stoke. Stereotactic transplantation of hADMSCs into peri-infarciton brain tissue can promote the post-infarction angiogenesis, but with less effects than those of VEGF. ObjectiveBased on the experiment results mentioned above, this study was designed to investigate the value of integrin-targeted angiogenesis imaging in stroke patients using a novel RGD-based imaging agents,68Ga-BNOTA-PRGD2(68Ga-PRGD2, for short).MethodsThis study had been approved by the Institutional Review Board of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences&Peking Union Medical College. All the patients signed the informed consent. A total of16stroke patients were enrolled (M11, F5,33-80y) and they received the brain68Ga-PRGD2PET/CT scan4days to13years after the event.68Ga-PRGD2was synthesized immediately before the injection with the radiochemical purity more than97%. Each patient was intravenously injected with the68Ga-PRGD2solution at a dose of approximately1.85MBq (0.05mCi) per kilogram body weight. Thirty minutes later, a brain scan (one10-minute bed position) was performed following a low-dose CT scan for attenuation correction and anatomic location.18F-FDG PET/CT study were performed within3days after the68Ga-PRGD2imaging.50min after intravenous injection of18F-FDG at a dosage of5.55 MBq (0.15mCi) per kilogram body weight, the low-dose CT scan and PET scanning were performed using the same scanning parameters as the68Ga-PRGD2PET/CT imaging. All patients underwent brain MRI which was performed conventionally in our hospital and eight patients had the MRI within3days. In addition, two patients also underwent repeat brain68Ga-PRGD2and18F-FDG PET/CT scans at3months after the event. The68Ga-PRGD2and18F-FDG PET/CT images, as well as the MIR images, were transferred to a workstation (MMWP, Siemens Medical Solution) for comparison and analysis. The co-registered and matched images were visually and semi-quantitatively analyzed. In semi-quantitative analysis, the peak standardized uptake values (pSUVs) of the68Ga-PRGD2accumulation were measured by obtaining the mean value of a region-of-interest (ROI) setting at the80%threshold of a lesion. The mean values of contralateral normal brain were measured as background and the lesion to background ratios were calculated. The maximum dimension (Dmax) of stroke was measured over the CT images by referring to the18F-FDG images. Correlation analysis between the pSUV of Ga-PRGD2uptake and the Dmax of stroke lesions was performed by bivariate correlation analysis using SPSS (version16.0, SPSS Inc., Chicago, IL). P<0.05was considered to be statistically significant.ResultsExcept for choroid plexus, normal brain was void of Ga-PRGD2uptake. By referring to the matched CT and18F-FDG images, as well as MRI in some cases, the Ga-PRGD2uptake was found at or around the stroke regions in eight of16stroke patients with pSUVs of0.46±0.29(0.15-0.93), which was significantly higher than the mean SUVs of the contralateral brain (0.15±0.09,0.05-0.30,.P＜0.001) and the lesion to contralateral background ratios were3.29±1.09(1.90-4.67). Among the eight patients without definite68Ga-PRGD2accumulation in the brain, four patients had suffered the event6months before, whereas the other four patients suffered slight event resulting from relatively small lesions in the basal ganglia (n=3) or in the corona radiate (n=1). In10stroke patients scanned13-26days after the event, pSUVs were significantly correlated with the maximum diameters of the stroke regions measured on CT (r=0.835, P=0.003). In addition, for the two follow-up patients, one still with significant symptoms had68Ga-PRGD2uptake, with the pSUV decreased from0.23to0.16, whereas the other patient who had good recovery experienced a disappearance of Ga-PRGD2accumulation.ConclusionThe avp3-integrin imaging method using68Ga-PRGD2PET/CT seems useful for evaluation of post-stroke repair by providing information related to angiogenesis. The success of this pilot study merits further clinical investigation of68Ga-PRGD2PET/CT in evaluation of post-stroke treatments.