Radiolabeled Risperidone microSPECT/CT Imaging for Intranasal Implant Studies Development.

The use of intranasal implantable drug delivery systems has many potential advantages for the treatment of different diseases, as they can provide sustained drug delivery, improving patient compliance. We describe a novel proof-of-concept methodological study using intranasal implants with radiolabeled risperidone (RISP) as a model molecule. This novel approach could provide very valuable data for the design and optimization of intranasal implants for sustained drug delivery. RISP was radiolabeled with 125I by solid supported direct halogen electrophilic substitution and added to a poly(lactide-co-glycolide) (PLGA; 75/25 D,L-Lactide/glycolide ratio) solution that was casted on top of 3D-printed silicone molds adapted for intranasal administration to laboratory animals. Implants were intranasally administered to rats, and radiolabeled RISP release followed for 4 weeks by in vivo non-invasive quantitative microSPECT/CT imaging. Percentage release data were compared with in vitro ones using radiolabeled implants containing either 125I-RISP or [125I]INa and also by HPLC measurement of drug release. Implants remained in the nasal cavity for up to a month and were slowly and steadily dissolved. All methods showed a fast release of the lipophilic drug in the first days with a steadier increase to reach a plateau after approximately 5 days. The release of [125I]I- took place at a much slower rate. We herein demonstrate the feasibility of this experimental approach to obtain high-resolution, non-invasive quantitative images of the release of the radiolabeled drug, providing valuable information for improved pharmaceutical development of intranasal implants.

Infection-specific PET imaging with 18F-fluorodeoxysorbitol and 2-[18F]F-ρ-aminobenzoic acid: An extended diagnostic tool for bacterial and fungal diseases.

Introduction: Suspected infectious diseases located in difficult-to-access sites can be challenging due to the need for invasive procedures to isolate the etiological agent. Positron emission tomography (PET) is a non-invasive imaging technology that can help locate the infection site. The most widely used radiotracer for PET imaging (2-deoxy-2[18F] fluoro-D-glucose: [18F]FDG) shows uptake in both infected and sterile inflammation. Therefore, there is a need to develop new radiotracers able to specifically detect microorganisms. Methods: We tested two specific radiotracers: 2-deoxy-2-[18F]-fluoro-D-sorbitol ([18F]FDS) and 2-[18F]F-ρ-aminobenzoic acid ([18F]FPABA), and also developed a simplified alternative of the latter for automated synthesis. Clinical and reference isolates of bacterial and yeast species (19 different strains in all) were tested in vitro and in an experimental mouse model of myositis infection. Results and discussion: Non-lactose fermenters (Pseudomonas aeruginosa and Stenotrophomonas maltophilia) were unable to take up [18F]FDG in vitro. [18F]FDS PET was able to visualize Enterobacterales myositis infection (i.e., Escherichia coli) and to differentiate between yeasts with differential assimilation of sorbitol (i.e., Candida albicans vs. Candida glabrata). All bacteria and yeasts tested were detected in vitro by [18F]FPABA. Furthermore, [18F]FPABA was able to distinguish between inflammation and infection in the myositis mouse model (E. coli and Staphylococcus aureus) and could be used as a probe for a wide variety of bacterial and fungal species.

Performance evaluation of a preclinical SPECT/CT system for multi-animal and multi-isotope quantitative experiments.

The aim was to study the performance of the U-SPECT6/CT E-class system for preclinical imaging, to later demonstrate the viability of simultaneous multi-animal and multi-isotope imaging with reliable quantitative accuracy. The performance of the SPECT was evaluated for two collimators dedicated for mouse (UHS-M) and rat imaging (UHR-RM) in terms of sensitivity, energy resolution, uniformity and spatial resolution. Point sources, hot­rod and uniform phantoms were scanned, and additional tests were carried out to evaluate singular settings such as simultaneous multi-isotope acquisition and imaging with a multi-bed system. For in-vivo evaluation, simultaneous triple-isotope and multi-animal studies were performed on mice. Sensitivity for 99mTc was 2370 cps/MBq for the UHS-M collimator and 493 cps/MBq for the UHR-RM. Rods of 0.6 mm and 0.9 mm were discernible with the UHS-M and UHR-RM collimators respectively, with optimized reconstruction. Uniformity in low counting conditions has proven to be poor (> 75%). Multi-isotope and multi-bed phantom acquisitions demonstrated accurate quantification. In mice, simultaneous multi-isotope imaging provided the separate distribution of 3 tracers and image quality of the multi-mouse bone scan was adequate. The U-SPECT6/CT E-class has shown good sensitivity and spatial resolution. This system provides quantitative images with suitable image quality for multi-mouse and multi-isotope acquisitions.

High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

Preclinical safety of negatively charged microspheres (NCMs): Optimization of radiolabeling for in vivo and ex vivo biodistribution studies after topical administration on full-thickness wounds in a rat model.

Negatively charged microspheres (NCMs) are postulated as a new form of treatment for chronic wounds. Despite the efficacy shown at clinical level, more studies are required to demonstrate their safety and local effect. The objective of the work was to confirm the lack of NCM systemic absorption performing a biodistribution study of the NCMs in an open wound rat animal model. To this end, radiolabeling of NCMs with technetium-99 m was optimized and biodistribution studies were performed by in vivo SPEC/CT imaging and ex vivo counting during 24 h after topical administration. The studies were performed on animals treated with a single or repeated dose to study the effect of macrophages during a prolonged treatment. NCM radiolabeling was achieved in a simple, efficient and stable manner with high yield. SPECT/CT images showed that almost all NCMs (about 85 %) remained on the wound for 24 h either after single or multiple administrations. Ex vivo biodistribution studies confirmed that there was no accumulation of NCMs in any organ or tissue except in the wound area, suggesting a lack of absorption. In conclusion, NCMs can be considered safe as local wound treatment since they remain at the administration area.

Cerebral metabolic pattern associated with progressive parkinsonism in non-human primates reveals early cortical hypometabolism.

Dopaminergic denervation in patients with Parkinson's disease is associated with changes in brain metabolism. Cerebral in-vivo mapping of glucose metabolism has been studied in severe stable parkinsonian monkeys, but data on brain metabolic changes in early stages of dopaminergic depletion of this model is lacking. Here, we report cerebral metabolic changes associated with progressive nigrostriatal lesion in the pre-symptomatic and symptomatic stages of the progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson's Disease. Monkeys (Macaca fascicularis) received MPTP injections biweekly to induce progressive grades of dopamine depletion. Monkeys were sorted according to motor scale assessments in control, asymptomatic, recovered, mild, and severe parkinsonian groups. Dopaminergic depletion in the striatum and cerebral metabolic patterns across groups were studied in vivo by positron emission tomography (PET) using monoaminergic ([11C]-dihydrotetrabenazine; 11C-DTBZ) and metabolic (2-[18F]-fluoro-2-deoxy-d-glucose; 18F-FDG) radiotracers. 11C-DTBZ-PET analysis showed progressive decrease of binding potential values in the striatum of monkeys throughout MPTP administration and the development of parkinsonian signs. 18F-FDG analysis in asymptomatic and recovered animals showed significant hypometabolism in temporal and parietal areas of the cerebral cortex in association with moderate dopaminergic nigrostriatal depletion. Cortical hypometabolism extended to involve a larger area in mild parkinsonian monkeys, which also exhibited hypermetabolism in the globus pallidum pars interna and cerebellum. In severe parkinsonian monkeys, cortical hypometabolism extended further to lateral-frontal cortices and hypermetabolism also ensued in the thalamus and cerebellum. Unbiased histological quantification of neurons in Brodmann's area 7 in the parietal cortex did not reveal neuron loss in parkinsonian monkeys versus controls. Early dopaminergic nigrostriatal depletion is associated with cortical, mainly temporo-parietal hypometabolism unrelated to neuron loss. These findings, together with recent evidence from Parkinson's Disease patients, suggest that early cortical hypometabolism may be associated and driven by subcortical changes that need to be evaluated appropriately. Altogether, these findings could be relevant when potential disease modifying therapies become available.

Id1 and PD-1 Combined Blockade Impairs Tumor Growth and Survival of KRAS-mutant Lung Cancer by Stimulating PD-L1 Expression and Tumor Infiltrating CD8+ T Cells.

The use of PD-1/PD-L1 checkpoint inhibitors in advanced NSCLC is associated with longer survival. However, many patients do not benefit from PD-1/PD-L1 blockade, largely because of immunosuppression. New immunotherapy-based combinations are under investigation in an attempt to improve outcomes. Id1 (inhibitor of differentiation 1) is involved in immunosuppression. In this study, we explored the potential synergistic effect of the combination of Id1 inhibition and pharmacological PD-L1 blockade in three different syngeneic murine KRAS-mutant lung adenocarcinoma models. TCGA analysis demonstrated a negative and statistically significant correlation between PD-L1 and Id1 expression levels. This observation was confirmed in vitro in human and murine KRAS-driven lung cancer cell lines. In vivo experiments in KRAS-mutant syngeneic and metastatic murine lung adenocarcinoma models showed that the combined blockade targeting Id1 and PD-1 was more effective than each treatment alone in terms of tumor growth impairment and overall survival improvement. Mechanistically, multiplex quantification of CD3+/CD4+/CD8+ T cells and flow cytometry analysis showed that combined therapy favors tumor infiltration by CD8+ T cells, whilst in vivo CD8+ T cell depletion led to tumor growth restoration. Co-culture assays using CD8+ cells and tumor cells showed that T cells present a higher antitumor effect when tumor cells lack Id1 expression. These findings highlight that Id1 blockade may contribute to a significant immune enhancement of antitumor efficacy of PD-1 inhibitors by increasing PD-L1 expression and harnessing tumor infiltration of CD8+ T lymphocytes.

Inhibitor of Differentiation-1 Sustains Mutant KRAS-Driven Progression, Maintenance, and Metastasis of Lung Adenocarcinoma via Regulation of a FOSL1 Network.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2-M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. SIGNIFICANCE: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Assessment of metabolic patterns and new antitumoral treatment in osteosarcoma xenograft models by [18F]FDG and sodium [18F]fluoride PET.

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. METHODS: Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. RESULTS: The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p < 0.001). Sensitivity for Na [18F] F was around 70% in both models, with a specificity of 100%. 531MII tumors showed a heterogeneous Na [18F] F uptake, significantly higher than 143B tumors (p < 0.01). Importantly, [18F] FDG and Na [18F] F uptake corresponded to highly cellular or osteoid-rich tumors in the histopathological analysis, respectively. [18F] FDG data confirmed that the oncolytic treatment of 531MII tumors produced a significant reduction in growth even with the 107 pfu dose. CONCLUSIONS: PET studies demonstrated that the different osteosarcoma xenograft models developed tumors with diverse metabolic patterns that can be described by multitracer PET studies. Since not all tumors produced abundant osteoid, [18F] FDG demonstrated a better sensitivity for tumor detection and was able to quantitatively monitor in vivo response to the oncolytic adenovirus VCN-01.

The inhibitor of differentiation-1 (Id1) enables lung cancer liver colonization through activation of an EMT program in tumor cells and establishment of the pre-metastatic niche.

Id1 promotes carcinogenesis and metastasis, and predicts prognosis of non-small cell lung cancer (NSCLC)-adenocarcionoma patients. We hypothesized that Id1 may play a critical role in lung cancer colonization of the liver by affecting both tumor cells and the microenvironment. Depleted levels of Id1 in LLC (Lewis lung carcinoma cells, LLC shId1) significantly reduced cell proliferation and migration in vitro. Genetic loss of Id1 in the host tissue (Id1-/- mice) impaired liver colonization and increased survival of Id1-/- animals. Histologically, the presence of Id1 in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1+/+ mice and Id1-/- mice injected with LLC control cells revealed that Id1 loss reduces the levels of EMT-related proteins, such as vimentin. In tissue microarrays containing 532 NSCLC patients' samples, we found that Id1 significantly correlated with vimentin and other EMT-related proteins. Id1 loss decreased the levels of vimentin, integrinß1, TGFß1 and snail, both in vitro and in vivo. Therefore, Id1 enables both LLC and the host microenvironment for an effective liver colonization, and may represent a novel therapeutic target to avoid NSCLC liver metastasis.

Non-invasive in vivo imaging of cardiac stem/progenitor cell biodistribution and retention after intracoronary and intramyocardial delivery in a swine model of chronic ischemia reperfusion injury.

BACKGROUND: The safety and efficacy of cardiac stem/progenitor cells (CSC) have been demonstrated in previous preclinical and clinical assays for heart failure. However, their optimal delivery route to the ischemic heart has not yet been assessed. This study was designed to determine by a non-invasive imaging technique (PET/CT) the biodistribution and acute retention of allogeneic pig CSC implanted by two different delivery routes, intracoronary (IC) and intramyocardial (IM), in a swine preclinical model of chronic ischemia-reperfusion. METHODS: Ischemia-reperfusion was induced in six Goettingen hybrid minipigs by 90 min coronary artery occlusion followed by reperfusion. Thirty days later, animals were allocated to receive IC (n = 3) or NOGA®-guided IM injection (n = 3) of 50 million of 18F-FDG/GFP-labeled allogeneic pig CSC. Acute retention was quantified by PET/CT 4 h after injection and cell engraftment assessed by immunohistochemical quantification of GFP+ cells three days post-injection. RESULTS: Biodistribution of 18F-FDG-labeled CSC was clearly visualized by PET/CT imaging and quantified. No statistical differences in acute cell retention (percentage of injected dose, %ID) were found in the heart when cells were administered by NOGA®-guided IM (13.4 ± 3.4%ID) or IC injections (17.4 ± 4.1%ID). Interestingly, engrafted CSC were histologically detected only after IM injection. CONCLUSION: PET/CT imaging of 18F-FDG-labeled CSC allows quantifying biodistribution and acute retention of implanted cells in a clinically relevant pig model of chronic myocardial infarction. Similar levels of acute retention are achieved when cells are IM or IC administered. However, acute cell retention does not correlate with cell engraftment, which is improved by IM injection.

Lessons from 11C-dihydrotetrabenazine imaging in a xenograft mouse model of rat insulinoma: is PET imaging of pancreatic beta cell mass feasible?

BACKGROUND: The feasibility of beta cell mass (BCM) imaging and quantification with positron emission tomography (PET) in the pancreas is controversial. In an effort to shed some light on this topic, we have used a xenograft model of rat insulinoma (RIN) in mice, mimicking an intramuscular islet transplantation situation. METHODS: A total of 105 RIN cells were subcutaneously implanted in nude mice (N.=8). Tumor size and glycaemia levels were determined daily. Rat C-peptide was measured to demonstrate rat insulin production. PET imaging with 11C-(+)-α-dihydrotetrabenazine (11C-DTBZ) was done at 3 and 4 weeks and compared with 18F-FDG and 18F-DOPA studies in the same mice. Ex-vivo autoradiography with 11C-DTBZ was carried out in frozen sections of tumors. VMAT2 expression was measured by Western-blot and immunohistochemistry in tumors and RIN cells. RESULTS: Functional rat insulin production in mice was demonstrated by substantial decrease in glycaemia (<50 mg/dL by week 4) and rat C-peptide levels (7.2±2.6 ng/mL) similar to those measured in control rats. PET studies showed that tumor imaging with 11C-DTBZ at four (N.=8) and five (N.=5) weeks was negative; only bigger tumors could be seen with 18F-DOPA. In explanted tumors 11C-DTBZ autoradiography was negative, albeit VMAT2 expression measured by Western-blot and immunohistochemistry was lower than in cultured RIN cells. CONCLUSIONS: Although insulinomas are fully functional it does not seem feasible to use 11C-DTBZ for in-vivo measuring of BCM. This might either be due to inherent technical limitations of PET, decrease in VMAT2 expression in the tumors due to unknown reasons, or other biological limiting facts.

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 Parkinson's 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.

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.

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 10(7) GFP-expressing fresh SVF cells or culture media as control. Heart function and tissue metabolism were determined by echocardiography and (18)F-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.

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.

Transplantation of mesenchymal stem cells exerts a greater long-term effect than bone marrow mononuclear cells in a chronic myocardial infarction model in rat.

The aim of this study is to assess the long-term effect of mesenchymal stem cells (MSC) transplantation in a rat model of chronic myocardial infarction (MI) in comparison with the effect of bone marrow mononuclear cells (BM-MNC) transplant. Five weeks after induction of MI, rats were allocated to receive intramyocardial injection of 10(6) GFP-expressing cells (BM-MNC or MSC) or medium as control. Heart function (echocardiography and (18)F-FDG-microPET) and histological studies were performed 3 months after transplantation and cell fate was analyzed along the experiment (1 and 2 weeks and 1 and 3 months). The main findings of this study were that both BM-derived populations, BM-MNC and MSC, induced a long-lasting (3 months) improvement in LVEF (BM-MNC: 26.61 +/- 2.01% to 46.61 +/- 3.7%, p < 0.05; MSC: 27.5 +/- 1.28% to 38.8 +/- 3.2%, p < 0.05) but remarkably, only MSC improved tissue metabolism quantified by (18)F-FDG uptake (71.15 +/- 1.27 to 76.31 +/- 1.11, p < 0.01), which was thereby associated with a smaller infarct size and scar collagen content and also with a higher revascularization degree. Altogether, results show that MSC provides a long-term superior benefit than whole BM-MNC transplantation in a rat model of chronic MI.

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction.

AIMS: To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction. METHODS: Ninety-five Sprague-Dawley rats underwent left coronary artery ligation and after 1 month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence. RESULTS: One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3+/-9.6% versus 27.7+/-8% pre-transplant) and tissue viability (64.78+/-7.2% versus 55.89+/-6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability. CONCLUSION: These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC.

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

UNLABELLED: 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 (13)N-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 (alpha-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 (13)N-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: (13)N-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.