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LABORATORY OF:"In Vivo" Molecular Imaging
CONTACT PERSON: Prof. Sambuceti Gianmario (University of Genova)
Phone +39 010 5552027 E-mail: Sambuceti@unige.it

Description of Laboratory and Expertise:

The main task of this lab is the development and the optimization of tools for "in vivo" cellular and molecular imaging. The lab is strictly connected with the Chair of Nuclear Medicine of the University of Genova and has already adapted radionuclide methods for studying trafficking and early kinetics of different cell types, in particular of endothelial progenitor cells, hematopoietic stem cells, mesenchymal stem cells, and dendritic cells. The lab takes the advantages of the availability of all the facilities of a complete and modern Nuclear Medicine Service such as an on site cyclotron and its related radiopharmacy, a PET/CT scanner and two single photon emission tomographic imaging systems.

Abstract of Activities:

We implemented in our lab a method for radioactive labelling and tracking of stem cells in intact animals to investigate the signalling between diseased tissues and stem cells. This approach has been applied to the study of endothelial progenitor cells (EPCs) in diabetes, demonstrating that this disease prolongs the persistence of EPCs in the blood, increases their lung sequestration and reduces their aortic uptake, accounting for the reduced expression of eNOS and CD31 of rats. The molecular mechanism behind this altered signalling can be largely and selectively counteracted by induction of heme oxygenase-1 expression.
Moreover, in collaboration with the Stem Cell Center of S. Martino Hospital (Drs. F. Frassoni and M. Podestà), we are now studying the kinetics of hematopoietic stem cells (HSC) in a model of bone marrow (BM) trasplantation. This program aims at identifying methods able to improve the seeding efficacy of cord blood transplant. In this line, intrabone (IB) injection of HSC has been proposed, improving HSC engraftment and reducing the incidence of graft-vs-host disease. We aim at exploring the early phase of HSC trafficking in order to evaluate the kinetics and the migration of these cells at the BM sites.
A parallel task of our group is the utilization of tracer kinetic methods to elucidate the mechanism of action of a surgical approach to type 2 diabetes (DM) proposed by a research team active in bariatric surgery at the University of Genova (Prof. N. Scopinaro). In this program, we aim at verifying the early and late effects of surgery on glucose metabolism in skeletal muscle, adipose tissue and myocardium, using dynamic PET scan, under fasting state and measuring tissue and whole body glucose consumption using the graphical Patlak approach. The preliminary data seem to suggest a differential effect of surgery on tissue metabolism, with a progressive increase of glucose uptake in myocardium, a biphasic response in skeletal muscle and no changes in the adipose tissue. So far, a software is under development in cooperation with the PET University Center in Turku Finnland, to analyse the response of liver metabolism to surgery.

Detailed Research Activities:

Despite an intense research activity, the therapeutic potential of stem cell (SC) in degenerative diseases has not been fully defined. These disappointing uncertainties are partially explained by the fact that the disease target of the intervention might impair the biology of SC and, mostly, the signalling events occurring in diseased tissues and affecting their recruitment. A better definition of this SC-target tissue interaction might improve the therapeutic effect of SC-based therapies. However, an integrated approach to the study of stem cell trafficking is needed to this purpose. In this lab, we implemented a method for radioactive labelling and tracking SC in intact animals. We used this tool to investigate, first, the kinetics of endothelial progenitor cells (EPCs) in a rat model of diabetes, as this condition accelerates atherosclerosis progression impairing both endothelial viability and SC biology via a common oxidative damage. We documented that normal EPCs show a prolonged persistence in blood, an increased lung sequestration and a reduced aortic uptake in diabetic rats that also showed an increased number of circulating endothelial cell fragments and a reduced expression of eNOS and CD31. Empowering the antioxidant potential (by induction of Hemeoxygenase-1 gene expression) largely prevented these alterations and, in parallel, improved EPC's recruitment by the whole body and by the aorta.
The development and optimization of a system for cell labelling and tracking has now been expanded to study the trafficking of different types of stem cells under different disease conditions. So far, an intense activity is in progress for the study of the kinetics of hematopoietic cells. In this setting, recent studies suggested a great potential of bone marrow transplantation using cord blood derived stem cells because of the relatively easy bankability of these cells. However, intravenous infusion of cord blood hematopoietic stem cells (HSC) suffers of a relatively low seeding efficency. To solve this problem, direct intrabone marrow (IBM) injection of HSC has been proposed by Dr. Francesco Frassoni and Colleagues at the Stem Cell Center of the University - San Martino Hospital in Genova. As a matter of fact, this route of administration has been shown to improve HSC engraftment and to reduce the incidence of graft-vs-host disease. Still, the mechanisms underlying this beneficial effect of intrabone injection and the implications of this administration route are largely unknown. In particular, whether the improved biology of intrabone administered HSC derives from an increased local engraftment or by an improved kinetics behavior is still largely unknown. The purpose of our study will be to explore the early phase of HSC trafficking in order to evaluate the kinetics and the migration of these cells at the BM sites and to verify whether IBM administered HSC will prove to be sensitive and adequate means to measure human HSC capability and enable us to investigate the interaction of HSC and bone marrow environment directly "in vivo".
Given the discovery that mesenchymal stem cells (MSCs) inhibit the proliferation of T lymphocytes, B lymphocytes and NK cells, and impair dendritic cells (DCs) maturation, the possibility to easily upgrade the above labelling tools to evaluate the trafficking of all immune system cells gives us the opportunity to further understand signalling mechanisms between MSCs and blood cells.
In particular , dendritic cells (DCs) are professional antigen-presenting cells (APC) endowed with the unique capacity to cluster naive T cells. Immature DCs process antigens and, in the presence of danger signals, enter a maturation process that modifies their functions, their response to chemokines and their chemokine secretion. Antigen presentation occurs in the regional lymph-nodes where DCs have moved to, under the influence of chemokines. Moreover, due to these properties, mature DCs are thought to be ideal for generating a primary immune response against cancer, viral infections, and other diseases. Currently, DCs loaded with tumor antigen-derived peptides are used in therapeutic vaccination in cancer patients. However, the optimal dose and route of administration of a DC vaccine still remain to be determined. To this aim, we are working on the optimization of a method for mature DCs labelling and "in vivo" monitoring under different activation conditions in mice. Once optimized, this method will give us the opportunity to test the effect of MSCs on DCs "in vivo" migration and to compare it with known activated or deactivated models.
In relation to the clinical exploitation of our methods, one of our major current programs is the characterization of type 2 diabetes (DM) as a major risk factor for cardiovascular events. Despite major advances in the therapeutic options for this syndrome, its prevalence is continuingly growing worldwide and this represents one of the possible explanations of the reduction in decrease of cardiovascular mortality observed in these last years. It is well known that type 2 DM is frequently associated with obesity and with the so called metabolic syndrome. A research team active in bariatric surgery for obese patients at our University (Prof. Nicola Scopinaro) reported since long time that interventions such as biliopancreatic diversion or gastric bypass are associated with an increased insulin sensitivity and substantially with the loss of need for antidiabetic agents, even in insulin-dependent patients. We took advantage of the cooperation with this group, and started a collaborative program aiming at verifying the early and late effects of surgery on glucose metabolism of skeletal muscle, adipose tissue and myocardium. To this purpose, we used a dynamic PET scan, under fasting state and measured tissue and whole body glucose consumption using the conventional Patlak approach. Preliminary data obtained in more than 20 patients seem to demonstrate a differential effect of surgery on tissue metabolism. These results have been communicated at the European Congress of Nuclear Medicine and document that glucose uptake progressively increases in the myocardium, has a biphasic response in the skeletal muscle and does not change at all in the adipose tissue. So far, a software is under development in cooperation with the PET University center in Turku (Finland), to analyse the response of liver metabolism to surgery.
Finally, in collaboration with the Vascular and Endovascular Surgery team of our University (Prof. Domenico Palombo), we are studing the potential of integrated positron emission tomography and computed tomography (PET/CT) to identify and quantify aneurysm wall inflammation with the aim to provide valuable "in vivo" information on the pathophysiological mechanisms leading to aneurysm formation and progression and to identify those patients who benefit from prophylactic aneurysm repair.
In fact, although surgical techniques and perioperative management improved dramatically over the past 50 years, patient selection is still based on measurement of aortic diameter. Some aneurysms, however,undergo rupture at an unexpectedly small size while other ones grow exceptionally large and remain intact. To decrease the number of unanticipated ruptures while reducing the number of unnecessary AAA repairs (and associated morbidity and mortality), a more accurate method for "in vivo" aneurysm rupture prediction is needed.
End-stage aneurysm disease and especially aneurysm rupture are characterized by extensive inflammation of the arterial wall. Although the stimulus for this enhanced infiltration is not known, recent insights into the pathophysiology of aneurysm formation, growth, and rupture indicate a close relationship between increased mechanical stress (hypertension) and the activation of infiltrated lymphocytes and macrophages. This increased inflammatory activity results in the progressive breakdown of the aortic wall, aneurysm dilatation, and, ultimately, rupture. As inflammation is characterized by increased metabolic activity of leukocytes, F18-fluorodeoxyglucose (FDG), a glucose derivative extensively used for positron emission tomography (PET) studies , shows increased uptake in inflammatory tissue.

Applications and Developments:

The "In Vivo" Molecular Imaging laboratory research activities may provide future applications and comebacks in the fields of hematopoietic stem cell kinetics and trafficking and of the development of cellular structures and chemical compounds or procedures favoring the "in vivo" mature bone marrow engraftment. These outcomes may be relevant for the area of cell-mediated tissue replacement. On the other hand, this laboratory aims at assessing the changes in glucose metabolism in diabetic patients, with particular attention to heart, muscle and liver.

Managed core facilities:

The lab takes the advantage of the facilities available at the nuclear medicine unit of the University Hospital San Martino. This unit is provided with an onsite cyclotron and its related radipharmacy lab, as well as with a high resolution PET/CT scanner. Moreover, within this unit there is a lab for cell culture, cell labelling and two systems for single photon tomographic acquisitions.

Ongoing collaborations:

A deep interaction is present between our center and the Genova Section of the CNR Institute of Bioimages and Molecular Physiology (Milan, Italy). The cooperation with the National Research Council is corroborated by the close links present so far with the CNR institute of Clinical Physiology in Pisa.
The study on the kinetics of endothelial progenitor cells is in progress as well as a cooperation with the New York Medical College and its Pharmacology Insttitute (prof. N Abraham)
Our research in the field of Stem Cells kinetics is going on in collaboration with the Centers of Stem Cell Center and of Nuclear Medicine of the University Hospital San Martino in Genova.
Research on the function of mesenchymal stem cells in neurologic disorders is going on in collaboration with the Neuroimmunobiology Laboratory of ABC (Dr. A. Uccelli).
Research on the assessment of glucose metabolism by 18F-FDG PET is going on in collaboration with the Department of Surgery of University - San Martino Hospital, while the studies on atherosclerotic aneurysms are performed in strict cooperation with the Unit of Vascular and Endovascular Surgery at ABC.

Most recent and significant publications:

Sambuceti G, Morbelli S, Vanella L, Kusmic C, Marini C, Massollo M, Augeri C, Corselli M, Ghersi C, Chiavarina B, Rodella LF, L'Abbate A, Drummond G, Abraham NG, Frassoni F. Diabetes Impairs Vascular Integrity and Recruitment of Normal Stem Cells by Oxidant Damage, Reversed by Increases in pAMPK, Heme Oxygenase-1 and Adiponectin. Stem Cells 2009; 27:399-407.

Kusmic C, Morbelli S, Marini C, Matteucci M, Cappellini C, Pomposelli E, Marzullo P, L'abbate A, Sambuceti G. Whole-body evaluation of MIBG tissue extraction in a mouse model of long-lasting type II diabetes and its relationship with norepinephrine transport protein concentration. J Nucl Med. 2008;49:1701-6.

Inglese E, Leva L, Matheoud R, Sacchetti G, Secco C, Gandolfo P, Brambilla M, Sambuceti G. Spatial and temporal heterogeneity of regional myocardial uptake in patients without heart disease under fasting conditions on repeated whole-body 18F-FDG PET/CT. J Nucl Med. 2007;48:1662-9.

Zacchigna S, Tasciotti E, Kusmic C, Arsic N, Sorace O, Marini C, Marzullo P, Pardini S, Petroni D, Pattarini L, Moimas S, Giacca M, Sambuceti G. In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature. Hum Gene Ther. 2007 Jun;18:515-24.

Marini C, Giorgetti A, Gimelli A, Kusch A, Sereni N, L'abbate A, Marzullo P, Sambuceti G. Extension of myocardial necrosis differently affects MIBG retention in heart failure caused by ischaemic heart disease or by dilated cardiomyopathy. Eur J Nucl Med Mol Imaging. 2005;32:682-8.

Sambuceti G, Marzilli M, Mari A, Marini C, Schluter M, Testa R, Papini M, Marraccini P, Ciriello G, Marzullo P, L'Abbate A. Coronary microcirculatory vasoconstriction is heterogeneously distributed in acutely ischemic myocardium. Am J Physiol Heart Circ Physiol. 2005;288:H2298-305

Morbelli S, Marini C, Bodrato S, Scopinaro N, Camerini G, Papadia F, Adami G, Sambuceti G. Quantitative 18F-FDG PET in defining benefit of biliopancreatic diversion in non obese patients with Type 2 diabetes mellitus. EANM 2008 (suppl)