Margarita Ecay

Adipose stromal vascular fraction improves cardiac function in chronic myocardial infarction through differentiation and paracrine activity.

Fresh adipose-derived cells have been shown to be effective in the treatment of acute myocardial infarction (MI), but their role in the chronic setting is unknown. We sought to determine the long-term effect of the adipose derived-stromal vascular fraction (SVF) cell transplantation in a rat model of chronic MI. MI was induced in 82 rats by permanent coronary artery ligation and 5 weeks later rats were allocated to receive an intramyocardial injection of 107 GFP-expressing fresh SVF cells or culture media as control. Heart function and tissue metabolism were determined by echocardiography and 18F-FDG-microPET, respectively, and histological studies were performed for up to 3 months after transplantation. SVF induced a statistically significant long-lasting (3 months) improvement in cardiac function and tissue metabolism that was associated with increased revascularization and positive heart remodeling, with a significantly smaller infarct size, thicker infarct wall, lower scar fibrosis, and lower cardiac hypertrophy. Importantly, injected cells engrafted and were detected in the treated hearts for at least 3 months, directly contributing to the vasculature and myofibroblasts and at negligible levels to cardiomyocytes. Furthermore, SVF release of angiogenic (VEGF and HGF) and proinflammatory (MCP-1) cytokines, as well as TIMP1 and TIMP4, was demonstrated in vitro and in vivo, strongly suggesting that they have a trophic effect. These results show the potential of SVF to contribute to the regeneration of ischemic tissue and to provide a long-term functional benefit in a rat model of chronic MI, by both direct and indirect mechanisms.

Twelve automated thresholding methods for segmentation of PET images: a phantom study.

Tumor volume delineation over positron emission tomography (PET) images is of great interest for proper diagnosis and therapy planning. However, standard segmentation techniques (manual or semi-automated) are operator dependent and time consuming while fully automated procedures are cumbersome or require complex mathematical development. The aim of this study was to segment PET images in a fully automated way by implementing a set of 12 automated thresholding algorithms, classical in the fields of optical character recognition, tissue engineering or non-destructive testing images in high-tech structures. Automated thresholding algorithms select a specific threshold for each image without any a priori spatial information of the segmented object or any special calibration of the tomograph, as opposed to usual thresholding methods for PET. Spherical (18)F-filled objects of different volumes were acquired on clinical PET/CT and on a small animal PET scanner, with three different signal-to-background ratios. Images were segmented with 12 automatic thresholding algorithms and results were compared with the standard segmentation reference, a threshold at 42% of the maximum uptake. Ridler and Ramesh thresholding algorithms based on clustering and histogram-shape information, respectively, provided better results that the classical 42%-based threshold (p < 0.05). We have herein demonstrated that fully automated thresholding algorithms can provide better results than classical PET segmentation tools.

MAPC transplantation confers a more durable benefit than AC133+ cell transplantation in severe hind limb ischemia.

There is a need for comparative studies to determine which cell types are better candidates to remedy ischemia. Here, we compared human AC133+ cells and multipotent adult progenitor cells (hMAPC) in a mouse model reminiscent of critical limb ischemia. hMAPC or hAC133+ cell transplantation induced a significant improvement in tissue perfusion (measured by microPET) 15 days posttransplantation compared to controls. This improvement persisted for 30 days in hMAPC-treated but not in hAC133+-injected animals. While transplantation of hAC133+ cells promoted capillary growth, hMAPC transplantation also induced collateral expansion, decreased muscle necrosis/fibrosis, and improved muscle regeneration. Incorporation of differentiated hAC133+ or hMAPC progeny into new vessels was limited; however, a paracrine angio/arteriogenic effect was demonstrated in animals treated with hMAPC. Accordingly, hMAPC-conditioned, but not hAC133+-conditioned, media stimulated vascular cell proliferation and prevented myoblast, endothelial, and smooth muscle cell apoptosis in vitro. Our study suggests that although hAC133+ cell and hMAPC transplantation both contribute to vascular regeneration in ischemic limbs, hMAPC exert a more robust effect through trophic mechanisms, which translated into collateral and muscle fiber regeneration. This, in turn, conferred tissue protection and regeneration with longer term functional improvement.

13N-ammonia PET as a measurement of hindlimb perfusion in a mouse model of peripheral artery occlusive disease

Peripheral arterial occlusive disease (PAOD) is a leading cause of mortality and morbidity in the western world. The development of noninvasive methods for assessment and comparison of the efficacy of novel therapies in animal models is of great importance. Methods: Hindlimb ischemia was induced in nude mice by ligation and excision of the left femoral artery (n = 5) or the left iliac artery (n = 10). Assessment of limb perfusion was performed by small-animal PET analysis after intravenous injection of 13N-ammonia between 24 h and 30 d after surgery using the ratio of perfusion between the left limb (ischemic) and the right limb (control). Activity concentration per area unit was calculated in regions of interest placed on 1-mm-thick images for numeric calculations, and the iliac and the femoral models were compared. In addition, histopathologic studies were performed to assess the degree of necrosis (hematoxylin−eosin) and fibrosis (sirius red). Immunohistochemistry analyses for identification of arterioles (α-smooth muscle actin) and endothelium—capillaries—(Bandeiraea simplicifolia I [BS-I] lectin) were also performed. Results: Perfusion in both hindlimbs of control animals was similar (median of the left-to-right ratio = 0.99). Twenty-four hours after ischemia, perfusion of the ischemic limb (% mean ± SD) was 33.3 ± 10.6 and 22.1 ± 9.9 in the femoral and iliac models, respectively. Spontaneous recovery of perfusion in the hindlimb that underwent surgery was significantly lower in the iliac model at day +15 (73.2 ± 15.5 vs. 51.9 ± 11.3; P < 0.01). Fibrosis increased progressively until day +30, whereas muscle necrosis was maximal at day +7 with a moderate reduction by day +30. In accordance with this positive effect, there was a statistically significant increase in the area covered with smooth muscle-coated vessels (arterioles) at day +30 in comparison with day 7 (P < 0.05). In addition, a correlation between 13N-ammonia uptake and the amount of necrosis (r = −0.73; P = 0.06) and fibrosis (r = −0.67; P = 0.05) at day +30 was found. Conclusion: 13N-Ammonia imaging allows semiquantitative evaluation of hindlimb perfusion in surgical mouse models of acute hindlimb ischemia. Although spontaneous perfusion recovery is observed in both models, the iliac model shows a substantially lower recovery and is hence better suited for assessment of new therapeutic strategies for acute hindlimb ischemic disease.

Monoaminergic PET imaging and histopathological correlation in unilateral and bilateral 6-hydroxydopamine lesioned rat models of Parkinson's disease: A longitudinal in-vivo study

Carbon-11 labeled dihydrotetrabenazine ((11)C-DTBZ) binds to the vesicular monoamine transporter 2 and has been used to assess nigro-striatal integrity in animal models and patients with Parkinsons disease. Here, we applied (11)C-DTBZ positron emission tomography (PET) to obtain longitudinally in-vivo assessment of striatal dopaminergic loss in the classic unilateral and in a novel bilateral 6-hydroxydopamine (6-OHDA) lesion rat model. Forty-four Sprague-Dawley rats were divided into 3 sub-groups: 1. 6-OHDA-induced unilateral lesion in the medial forebrain bundle, 2. bilateral lesion by injection of 6-OHDA in the third ventricle, and 3. vehicle injection in either site. (11)C-DTBZ PET studies were investigated in the same animals successively at baseline, 1, 3 and 6weeks after lesion using an anatomically standardized volumes-of-interest approach. Additionally, 12 rats had PET and Magnetic Resonance Imaging to construct a new (11)C-DTBZ PET template. Behavior was characterized by rotational, catalepsy and limb-use asymmetry tests and dopaminergic striatal denervation was validated post-mortem by immunostaining of the dopamine transporter (DAT). (11)C-DTBZ PET showed a significant decrease of striatal binding (SB) values one week after the unilateral lesion. At this point, there was a 60% reduction in SB in the affected hemisphere compared with baseline values in 6-OHDA unilaterally lesioned animals. A 46% symmetric reduction over baseline SB values was found in bilaterally lesioned rats at the first week after lesion. SB values remained constant in unilaterally lesioned rats whereas animals with bilateral lesions showed a modest (22%) increase in binding values at the 3rd and 6th weeks post-lesion. The degree of striatal dopaminergic denervation was corroborated histologically by DAT immunostaining. Statistical analysis revealed a high correlation between (11)C-DTBZ PET SB and striatal DAT immunostaining values (r=0.95, p<0.001). The data presented here indicate that (11)C-DTBZ PET may be used to ascertain changes occurring in-vivo throughout the evolution of nigro-striatal dopaminergic neurodegeneration, mainly in the unilateral 6-OHDA lesion rat.

Abstract 1996: Inhibitor of differentiation-1 (Id1) expression deficiency in the tumor microenvironment impairs experimental hepatic metastasis of lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Liver metastasis (LM) occurs in 30% of non-small cell lung cancer patients but the contribution of the patients genetic background to the hepatic metastasis development is unclear. Previously, we reported that inflammation contributes to the prometastatic microenvironment of the liver. Upregulation of transcriptional regulator Id1 gene has been associated with inflammation while ablation of Id1 in mice reduced inflammation. In this study Id1 deficient mice were used to analyze the role of host Id1 in the hepatic colonization of an experimental lung cancer. Methods: Lewis lung carcinoma cells were used for the experimental production of hepatic metastasis via intrasplenic injection of cancer cells in C57BL/6 wild-type and Id1-knockout (KO) mice. Animals were splenectomized to avoid flank tumor formation and weekly FDG-micro-positron emission tomographies (PET) were performed to monitor LM formation. Animals were sacrificed at the time of LM occurrence and total RNA was purified from LM. A microarray gene expression analysis (Affymetrix) with the support of Ingenuity Pathways Analysis (IPA) was performed to evaluate the potential impact of Id1 deficiency on the regulation of genes mediating cancer cell invasion and proliferation, angiogenesis and metastasis. Results: By week two after cancer cell injection, 70% wild-type and 10% Id1-KO mice had detectable hepatic metastasis by FDG-PET (p=0.02). Three weeks after injection, when 100% wild-type had LM, still just 20% Id1-KO mice had LM (p=0.015). Moreover, 50% Id1-KO mice remained LM-free >4 weeks after cancer cell injection. No other metastasis sites were detected at necropsy. A microarray gene expression analysis of LM from Id1-KO animals uncovered a remarkable downregulation (p<0.05) of specific genes involved in the activation of cancer cell proliferation (Myc, Cdc20, Smc2, Aurora kinase B, Cyclin B1, CDK1, TIMP1, Epiregulin), migration (Ccl7, Serpine1/PAI-1, VIM, Anxa2, S100A4, S100A6, Akt3, Adrenomedullin), angiogenesis (Hif1a, PGF, Nestin) and metastasis (FGFR1, Src, IL-18, MMP3, MMP12, MMP13, Amphiregulin, PDGFA, FoxM1, Hsp90AA1, MIF, Ccl2, RhoC). Conclusion: Our results demonstrate that Id1 expression deficiency impaired the metastatic process of lung cancer cells to the liver through the specific downregulation of key metastasis-associated genes and suggest that Id1-dependent mechanisms are new targets for hepatic metastasis therapeutic. This study has been partially funded by “UTE project CIMA” and an ISCIII-FIS grant 2011. Citation Format: Ignacio Gil-Bazo, Eduardo Castanon, Ines Lopez, Victor Segura, Mariano Ponz-Sarvise, Jose M. Lopez-Picazo, Maria Collantes, Margarita Ecay, Isabel Gil-Aldea, Alfonso Calvo, Fernando Vidal-Vanaclocha. Inhibitor of differentiation-1 (Id1) expression deficiency in the tumor microenvironment impairs experimental hepatic metastasis of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2014-1996