RESUMO
We combined multimodal imaging (bioluminescence, X-ray computed tomography, and PET), tomographic reconstruction of bioluminescent sources, and two unique, complementary models to evaluate three previously synthesized PET radiotracers thought to target pancreatic beta cells. The three radiotracers {[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]FP-DTBZ), [(18)F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline ((18)F-AV-266), and (2S,3R,11bR)-9-(3-fluoropropoxy)-2-(hydroxymethyl)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol ((18)F-AV-300)} bind vesicular monoamine transporter 2. Tomographic reconstruction of the bioluminescent signal in mice expressing luciferase only in pancreatic beta cells was used to delineate the pancreas and was coregistered with PET and X-ray computed tomography images. This strategy enabled unambiguous identification of the pancreas on PET images, permitting accurate quantification of the pancreatic PET signal. We show here that, after conditional, specific, and rapid mouse beta-cell ablation, beta-cell loss was detected by bioluminescence imaging but not by PET imaging, given that the pancreatic signal provided by three PET radiotracers was not altered. To determine whether these ligands bound human beta cells in vivo, we imaged mice transplanted with luciferase-expressing human islets. The human islets were imaged by bioluminescence but not with the PET ligands, indicating that these vesicular monoamine transporter 2-directed ligands did not specifically bind beta cells. These data demonstrate the utility of coregistered multimodal imaging as a platform for evaluation and validation of candidate ligands for imaging islets.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Diabetes Mellitus/metabolismo , Diagnóstico por Imagem/métodos , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ligantes , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos NOD , Ratos , Distribuição TecidualRESUMO
There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the ß cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and ß cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and ß cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in ß cell proliferation and ß cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in ß cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in ß cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs ß cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.
Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Contagem de Células , Células Endoteliais/metabolismo , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To survive and function, transplanted islets must revascularize because islet isolation severs arterial and venous connections; the current paradigm is that islet revascularization originates from the transplant recipient. Because isolated islets retain intraislet endothelial cells, we determined whether these endothelial cells contribute to the revascularization using a murine model with tagged endothelial cells (lacZ knock-in to Flk-1/VEGFR2 gene) and using transplanted human islets. At 3-5 weeks after transplantation beneath the renal capsule, we found that islets were revascularized and that the transplant recipient vasculature indeed contributed to the revascularization process. Using the lacZ-tagged endothelial cell model, we found that intraislet endothelial cells not only survived after transplantation but became a functional part of revascularized islet graft. A similar contribution of intraislet endothelial cells was also seen with human islets transplanted into an immunodeficient mouse model. In the murine model, individual blood vessels within the islet graft consisted of donor or recipient endothelial cells or were a chimera of donor and recipient endothelial cells, indicating that both sources of endothelial cells contribute to the new vasculature. These observations suggest that interventions to activate, amplify, or sustain intraislet endothelial cells before and after transplantation may facilitate islet revascularization, enhance islet survival, and improve islet transplantation.
Assuntos
Endotélio Vascular/fisiopatologia , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica , Animais , Animais Geneticamente Modificados , Biomarcadores/análise , Sobrevivência Celular , Endotélio Vascular/patologia , Humanos , Técnicas In Vitro , Ilhotas Pancreáticas/metabolismo , Rim/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Pâncreas/metabolismo , Transplante Heterólogo , Transplante Heterotópico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Pancreatic islet transplantation is an emerging therapy for type 1 diabetes, but it is difficult to assess islets after transplantation and thus to design interventions to improve islet survival. METHODS: To image and quantify islets, the authors transplanted luciferase-expressing murine or human islets (by adenovirus-mediated gene transfer) into the liver or beneath the renal capsule of immunodeficient mice and quantified the in vivo bioluminescence imaging (BLI) of mice using a cooled charge-coupled device camera and digital photon-counting image analysis. To account for variables that are independent of islet mass such as transplant site, animal positioning, and wound healing, the BLI of transplanted islets was calibrated against measurement of luminescence of an implanted bead emitting a constant light intensity. RESULTS: BLI of mice bearing islet transplants was seen in the expected anatomic location, was stable for more than 8 weeks after transplantation, and correlated with the number of islets transplanted into the liver or kidney. BLI of the luminescent bead and of transplanted islets in the kidney was approximately four times greater than when transplanted in the liver, indicating that photon emission is dependent on optical absorption of generated light and thus light source location. CONCLUSION: In vivo BLI allows for quantitative, serial measurements of pancreatic islet mass after transplantation and should be useful in assessing interventions to sustain or increase islet survival of transplanted islets.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/citologia , Animais , Sobrevivência de Enxerto , Humanos , Imageamento Tridimensional , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Medições Luminescentes , CamundongosRESUMO
PURPOSE: We developed a mouse model that enables non-invasive assessment of changes in beta cell mass. PROCEDURES: We generated a transgenic mouse expressing luciferase under control of the mouse insulin I promoter [mouse insulin promoter-luciferase-Vanderbilt University (MIP-Luc-VU)] and characterized this model in mice with increased or decreased beta cell mass and after islet transplantation. RESULTS: Streptozotocin-induced, diabetic MIP-Luc-VU mice had a progressive decline in bioluminescence that correlated with a decrease in beta cell mass. MIP-Luc-VU animals fed a high-fat diet displayed a progressive increase in bioluminescence that reflected an increase in beta cell mass. MIP-Luc-VU islets transplanted beneath the renal capsule or into the liver emitted bioluminescence proportional to the number of islets transplanted and could be imaged for more than a year. CONCLUSIONS: Bioluminescence in the MIP-Luc-VU mouse model is proportional to beta cell mass in the setting of increased and decreased beta cell mass and after transplantation.
Assuntos
Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas , Medições Luminescentes/métodos , Imagem Molecular/métodos , Animais , Tamanho Celular , Teste de Tolerância a Glucose , Insulina/genética , Luciferases/metabolismo , Camundongos , Modelos Animais , Regiões Promotoras Genéticas/genética , Imagem Corporal TotalRESUMO
In type 2 diabetes mellitus, insulin resistance and an inadequate pancreatic beta-cell response to the demands of insulin resistance lead to impaired insulin secretion and hyperglycemia. Pancreatic duodenal homeodomain-1 (PDX-1), a transcription factor required for normal pancreatic development, also plays a key role in normal insulin secretion by islets. To investigate the role of PDX-1 in islet compensation for insulin resistance, we examined glucose disposal, insulin secretion, and islet cell mass in mice of four different genotypes: wild-type mice, mice with one PDX-1 allele inactivated (PDX-1+/-, resulting in impaired insulin secretion), mice with one GLUT4 allele inactivated (GLUT4+/-, resulting in insulin resistance), and mice heterozygous for both PDX-1 and GLUT4 (GLUT4+/-;PDX-1+/-). The combination of PDX-1 and GLUT4 heterozygosity markedly prolonged glucose clearance. GLUT4+/-;PDX-1+/- mice developed beta-cell hyperplasia but failed to increase their beta-cell insulin content. These results indicate that PDX-1 heterozygosity (approximately 60% of normal protein levels) abrogates the beta-cell's compensatory response to insulin resistance, impairs glucose homeostasis, and may contribute to the pathogenesis of type 2 diabetes.