Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells

Aims Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. Methods and results MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. Conclusion MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulatio

Non-invasive stem cells imaging with animal PET after transplantation into the heart experimental validation

Ischemic heart disease (IHD) is a major cause of morbidity and mortality in most industrialized countries. Although therapies are available to delay disease progression,these approaches are unable to reverse or prevent the loss of cardiomyocytes that accompanies IHD. In the early 2000s, a novel regenerative therapy using stem cells (SCs) emerged as a promising alternative treatment for IHD. However, to date, results obtained from multiple preclinical and clinical studies have shown only a moderate efficacy for this treatment approach, and many questions remain. In this context, the development of a noninvasive imaging modality able to track SCs after they are delivered into the myocardium may help researchers to define the optimal cell type and delivery method, as well as to evaluate strategies for improving SC survival and/or engraftment after transplantation. Therefore, the objective of this work was to develop such a non-invasive imaging approach for the purpose of SC monitoring in small and large animals, using positron emission tomography (PET). The reporter gene herpes simplex type 1 thymidine kinase (HSV1-tk) or its mutant (HSV1-sr39tk) was introduced into mesenchymal stem cells (MSCs) by using adenoviral or retroviral vectors. The corresponding enzyme of HSV1-TK and HSV1-sr39TK phosphorylates the reporter probes, pyrimidine nucleoside derivatives and acycloguanosine derivatives (F-18-FHBG [9-(4-F-18-fluoro-3-[hydroxymethyl]butyl)guanine] for imaging and H-3-PCV [H-3-Penciclovir] for in vitro experiments). These probes become negatively charged after phosphorylation and are unable to cross the cell membrane. The intracellularly trapped radioactivity emits a signal that can be detected and quantified with an appropriate detector. A direct comparison of two types of viral vectors for transduction of the HSV1-tk gene revealed that both adenoviruses and retroviruses expressing the HSV1-tk gene could transduce rat MSCs without altering the cell phenotype, viability,proliferation rate, or differentiation capabilities. After they were delivered into a peripheral muscle of living rats, MSCs transduced with either virus were noninvasively imaged by F-18-FHBG and small-animal PET. The activity measured by PET at the injection site correlated well with the number of injected cells. As expected, retroviral transduction of MSCs allowed transplanted cells to be tracked for a longer duration than adenoviral transduction (up to 2 months vs. a few days after transduction, respectively). We tested the ability of the developed technique to image MSCs or Sca-1+ cardiac stem cells (CSCs) after their delivery into the rat or mouse myocardium, respectively. We developed a high-resolution ultrasound-guided technique to deliver SCs into the rodent myocardium. Sca-1+ CSCs were successfully transduced with a retrovirus carrying the HSV1-tk reporter gene without alteration of their phenotype, viability, or proliferation rate. Transduced MSCs and Sca-1+ CSCs were successfully imaged by F-18-FHBG and small-animal PET after their delivery into the rat and mouse myocardium, respectively, under echographic guidance. We also imaged transduced MSCs after their delivery into the pig myocardium by using a clinical PET-CT scanner. This experiment showed our ability to translate the reporter gene imaging approach from small to large animals. Lastly, we developed an original algorithm to quantify the infarct size in mice by myocardial perfusion SPECT, without the need of a normal perfusion database.Les cardiopathies d’origine ischémiques sont la principale cause de morbidité et de mortalité dans la plupart des pays développés. Les traitements standard actuels parviennent à réduire la sévérité des symptômes et ralentissent la progression vers l’insuffisance cardiaque mais n’ont pas d’impact sur le déficit en cellules cardiaques fonctionnelles. Depuis un peu plus d’une dizaine d’années, la thérapie cellulaire s’est imposée comme une nouvelle option possible pour améliorer le traitement de l’infarctus du myocarde et de l’insuffisance cardiaque chronique. Cependant, les multiples travaux expérimentaux et les nombreuses études cliniques realisés à ce jour ont rapporté une efficacité modérée et de nombreuses questions doivent encore être résolues afin de pouvoir optimiser cette nouvelle approche thérapeutique. Dans ce contexte, posséder une technique d’imagerie non-invasive capable de suivre in vivo le devenir de cellules souches transplantées au niveau du myocarde pourrait contribuer à répondre à certaines de ces questions. En effet, une telle technique pourrait permettre de mieux évaluer le type optimal de cellules à utiliser, la meilleure voie d’administration ou encore de nouvelles stratégies permettant d’améliorer la survie ou l’implantation des cellules. Par conséquent, le but de ce projet a été de mettre au point une technique d’imagerie non-invasive permettant de détecter, de localiser et de suivre le devenir de cellules souches transplantées dans le muscle cardiaque, tant de petits que de gros animaux. La technique développée utilise la tomographie par émission de positons (TEP) et les cellules ont été marquées au moyen du gène rapporteur de la thymidine kinase du virus de l’herpès simplex de type 1 (HSV1-tk) ou son mutant (HSV1-sr39tk). Les cellules souches mésenchymateuses (CSMs) ont été le type cellulaire initialement utilisé pour la mise au point. Le principe de l’utilisation du gène rapporteur HSV1-tk est le suivant: le gène rapporteur HSV1-tk ou son mutant est introduit dans des CSMs par transduction adénovirale ou rétrovirale. Leurs enzymes respectifs catalysent la phosphorylation de dérivés pyrimidiques et puriques ainsi que leurs analogues marqués (tels que le [F-18]-FHBG ou [F-18]-fluorohydroxymethylbutylguanine [pour l’imagerie] ou le H-3-PCV ou Penciclovir tritié [pour les expériences in vitro]). Une fois phosphorylés, ces dérivés s’accumulent à l’intérieur des cellules car les transporteurs membranaires ne savent pas les en expulser. Le signal émis par le traceur peut être enregistré et quantifié au moyen d’un détecteur approprié. Le premier objectif du travail a été de comparer l’effet de l’utilisation d’un vecteur adenoviral à l’utilisation d’un vecteur retroviral sur l’expression du HSV1- tk par les CSMs. Nous avons observé que ni la transduction adénovirale ni la transduction rétrovirale ne modifie le phénotype, la prolifération ou la capacité de différentiation des CSMs. En outre, les CSMs transduitess avec les deux types de vecteurs viraux peuvent être imagées de manière non-invasive au moyen d’une caméra TEP et de F-18-FHBG après transplantation dans un muscle périphérique de rat. Les images obtenues montrent une activité au niveau du site d’injection des cellules qui est proportionnelle au nombre de cellules transplantées. Comme attendu, l’expression du gène de la thymidine kinase par les CSMs dure nettement plus longtemps après transduction rétrovirale qu’après transduction adénovirale (2 mois versus quelques jours, respectivement). Ensuite, nous avons utilisé la technique mise au point pour imager des CSMs transplantées au niveau du myocarde de rat ainsi que pour imager des cellules souches cardiaques de souris Sca-1 positives (Sca-1+ CSCs) transplantées dans le myocarde de souris. Pour ce faire, nous avons d’abord développé une technique d’injection intramyocardique sous guidage échogaphique (à thorax fermé). Ensuite, nous avons montré que l’infection rétrovirale de Sca-1+ CSCs n’altérait ni le phénotype, ni la viabilité, ni la capacité de proliférer de ces cellules. Finalement,nous avons montré que les cellules MSCs et Sca-1+ CSCs infectées pouvaient être imagées au moyen de la TEP et de F-18-FHBG après transplantation dans le myocarde de rat ou de souris sous guidage échographique. En outre, en réussissant à imager au moyen d’une caméra PET-CT utilisée en clinique des CSMs implantées par voie trans-coronarienne dans du myocarde de cochon, nous avons montré notre capacité à pouvoir utiliser cette technique d’imagerie pour des expériences sur de « gros animaux ». Finalement, nous avons développé un algorithme original permettant de calculer la taille d’un infarctus sur des images SPECT de perfusion myocardique réalisées chez la souris, sans devoir recourir à l’utilisation d’une banque de données d'animaux normaux.(MED 3) -- UCL, 201

Iodine-131-MIBG scintigraphy in adults: interpretation revisited?

Iodine-131-metaiodobenzylguanidine (MIBG) scintigraphy is a reliable method used to diagnose pheochromocytoma. Although the adrenal medulla usually is not visualized, faint uptake can be observed in 16% of the patients 48-72 hr after injection of 18.5-37 MBq 131I-MIBG. We recently observed an increase in the frequency of visualization of the adrenal medulla in patients injected with 74 MBq 131I-MIBG. Therefore, we retrospectively evaluated the pattern of uptake and potential changes between 1984 and 1994. METHODS: Scintigraphic data from 103 patients referred for suspected pheochromocytoma were reviewed randomly. Data from 19 patients with medullary thyroid carcinoma were analyzed separately. Patients were injected with 74 MBq 131I-MIBG and imaged at 24 hr postinjection, 48 hr postinjection, or both. Adrenal uptake was scored visually as 0 (no visible uptake) and 1 (uptake just visible) to 4 (most intense activity in the picture). Semiquantitative indicies were evaluated for discriminating between normal adrenal medullae and pheochromocytomas. Twenty-seven pheochromocytomas were surgically proven in 25 patients. RESULTS: A visual score > or =3 was noted in 81% and 90% of the pheochromocytomas at 24 hr and 48 hr postinjection, respectively. From 1984 to 1988, 16% and 31% of adrenal medullae were seen at 24 and 48 hr postinjection, respectively, whereas from 1989 to 1994, 56% and 73% were visualized at 24 and 48 hr postinjection, respectively. Before 1989, the best cutoff criterion to identify a pheochromocytoma, determined from receiver operating characteristic curve analysis, was a score > or =1 at 24 hr and > or =3 at 48 hr postinjection, with a sensitivity and specificity of 92% and 84% at 24 hr and 92% and 99% at 48 hr postinjection. From 1989, the best cutoff was a score > or =3 at both imaging sessions, with a sensitivity and specificity of 82% and 100% at 24 hr and 100% and 97% at 48 hr postinjection. Among the semiquantitative indicies, the adrenal-to-liver and adrenal-to-heart ratios were the best discriminators between normal and pathological adrenals. They were, however, of little use because of the overlap between normal adrenal medullae and pheochromocytomas. CONCLUSION: The high rate of visualization of the normal adrenal medulla in this study was related to the larger-than-usual injected dose (74 MBq). Over recent years, however, this rate has been increasing, possibly because of the increased specific activity of 31I-MIBG. Adequate interpretation should take into account that a faint or definite uptake may be visible in more than 50% of normal adrenal medullae

Cardiac atrial metabolism quantitative assessment with analog and digital time of flight-PET/computed tomography.

Evaluation of left atrium (LA) remodeling is becoming increasingly relevant in understanding several pathological cardiac conditions. 18F-FDG-PET/computed tomography (CT), the current gold standard for metabolic evaluation of the left ventricle, could be extended to LA using the latest PET technologies. We sought to perform a phantom study to optimize the reconstruction algorithm in this context. The liver, heart cavity and walls of an anthropomorphic phantom were filled with typical patient 18F-FDG activity concentrations. Acquisitions were performed on an analog and on a digital TOF-PET/CT, and reconstructed with and without resolution recovery (RR). The Richardson-Lucy RR method was used, either through a third-party software or through the PET/CT manufacturer algorithm. Activity recoveries in the atria and ventricles and signal-to-noise ratios were evaluated to identify the best reconstruction and RR parameters. The same methodology was applied on a patient cardiac study. Analog PET/CT with the third-party RR cannot improve the activity recovery without markedly degrading the image quality. For the digital PET/CT, the optimal algorithm was the manufacturer RR reconstruction using four iterations and 15 subsets combined with five RR iterations. This reconstruction improved the LA activity recovery from 58% to 70% while preserving images of diagnostic quality. Similar results were obtained for the patient study. The digital TOF-PET/CT with the identified optimal reconstruction can be used to quantitatively analyze the LA uptake in 18F-FDG cardiac studies while still preserving image reading quality. This may lead to more precise cardiovascular disease status evaluation, especially when atria are concerned

Myocardial infarct size quantification in mice by SPECT using a novel algorithm independent of a normal perfusion database

Background: There is a growing interest in developing non-invasive imaging techniques permitting infarct size (IS) measurements in mice. The aim of this study was to validate the high-resolution rodent Linoview single photon emission computed tomography (SPECT) system for non-invasive measurements of IS in mice by using a novel algorithm independent of a normal database, in comparison with histology. Methods: Eleven mice underwent a left coronary artery ligature. Seven days later, animals were imaged on the SPECT 2h30 after injection of 173 ± 27 MBq of Tc-99m-sestamibi. Mice were subsequently killed, and their hearts were excised for IS determination with triphenyltetrazolium chloride (TTC) staining. SPECT images were reconstructed using the expectation maximization maximum likelihood algorithm, and the IS was calculated using a novel algorithm applied on the 20-segment polar map provided by the commercially available QPS software (Cedars-Sinai Medical Center, CA, USA). This original method is attractive by the fact that it does not require the implementation of a normal perfusion database. Results: Reconstructed images allowed a clear delineation of the left ventricles borders in all mice. No significant difference was found between mean IS determined by SPECT and by TTC staining [37.9 ± 17.5% vs 35.6 ± 17.2%, respectively (P = 0.10)]. Linear regression analysis showed an excellent correlation between IS measured on the SPECT images and IS obtained with TTC staining (y = 0.95x + 0.03 (r = 0.97; P < 0.0001)), without bias, as demonstrated by the Bland-Altman plot. Conclusion: Our results demonstrate the accuracy of the method for the measurement of myocardial IS in mice with the Linoview SPECT system