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1.
Stem Cell Res ; 78: 103462, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38851031

ABSTRACT

Spinocerebellar Ataxia Type 7 (SCA7) is an autosomal dominantly inherited disorder, primarily characterized by cerebellar ataxia and visual loss. SCA7 is caused by a CAG repeat expansion in exon 3 of the ATXN7 gene. We generated human induced pluripotent stem cells (hiPSCs) from peripheral blood-derived erythroblasts from two SCA7 patients (LUMCi051-A,B and LUMCi052-A,B,C) using integration-free episomal vectors. All hiPSC clones express pluripotency factors, show a normal karyotype, and can differentiate into the three germ layers. These lines can be used for in vitro disease modeling and therapy testing.


Subject(s)
Induced Pluripotent Stem Cells , Spinocerebellar Ataxias , Humans , Induced Pluripotent Stem Cells/metabolism , Spinocerebellar Ataxias/pathology , Spinocerebellar Ataxias/genetics , Cell Line , Male , Cell Differentiation , Female , Adult
2.
Mov Disord ; 38(8): 1428-1442, 2023 08.
Article in English | MEDLINE | ID: mdl-37278528

ABSTRACT

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein resulting in neuropathology including mutant ataxin-1 protein aggregation, aberrant neurodevelopment, and mitochondrial dysfunction. OBJECTIVES: Identify SCA1-relevant phenotypes in patient-specific fibroblasts and SCA1 induced pluripotent stem cells (iPSCs) neuronal cultures. METHODS: SCA1 iPSCs were generated and differentiated into neuronal cultures. Protein aggregation and neuronal morphology were evaluated using fluorescent microscopy. Mitochondrial respiration was measured using the Seahorse Analyzer. The multi-electrode array (MEA) was used to identify network activity. Finally, gene expression changes were studied using RNA-seq to identify disease-specific mechanisms. RESULTS: Bioenergetics deficits in patient-derived fibroblasts and SCA1 neuronal cultures showed altered oxygen consumption rate, suggesting involvement of mitochondrial dysfunction in SCA1. In SCA1 hiPSC-derived neuronal cells, nuclear and cytoplasmic aggregates were identified similar in localization as aggregates in SCA1 postmortem brain tissue. SCA1 hiPSC-derived neuronal cells showed reduced dendrite length and number of branching points while MEA recordings identified delayed development in network activity in SCA1 hiPSC-derived neuronal cells. Transcriptome analysis identified 1050 differentially expressed genes in SCA1 hiPSC-derived neuronal cells associated with synapse organization and neuron projection guidance, where a subgroup of 151 genes was highly associated with SCA1 phenotypes and linked to SCA1 relevant signaling pathways. CONCLUSIONS: Patient-derived cells recapitulate key pathological features of SCA1 pathogenesis providing a valuable tool for the identification of novel disease-specific processes. This model can be used for high throughput screenings to identify compounds, which may prevent or rescue neurodegeneration in this devastating disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Subject(s)
Induced Pluripotent Stem Cells , Spinocerebellar Ataxias , Mice , Animals , Ataxins/metabolism , Protein Aggregates , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Mice, Transgenic , Purkinje Cells/metabolism , Purkinje Cells/pathology , Spinocerebellar Ataxias/metabolism , Fibroblasts/metabolism
3.
Front Aging Neurosci ; 14: 1048584, 2022.
Article in English | MEDLINE | ID: mdl-36733499

ABSTRACT

Introduction: ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (Aß) domain of APP and leads to Aß peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model. Methods: We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110). Results: D-CAA cerebral organoids exhibited Aß accumulations, showed enhanced neuronal and astrocytic gene expression and TGFß pathway de-regulation. Conclusions: These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various Aß-related disorders.

4.
Nucleic Acid Ther ; 31(5): 351-363, 2021 10.
Article in English | MEDLINE | ID: mdl-34061681

ABSTRACT

Dutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon 17 of the amyloid precursor protein (APP) gene that leads to an amino acid substitution at codon 693. The mutation is located within the amyloid beta (Aß) domain of APP, and leads to accumulation of toxic Aß peptide in and around the cerebral vasculature. We have designed an antisense oligonucleotide (AON) approach that results in skipping of exon 17, generating a shorter APP isoform that lacks part of the Aß domain and the D-CAA mutation. We demonstrate efficient AON-induced skipping of exon 17 at RNA level and the occurrence of a shorter APP protein isoform in three different cell types. This resulted in a reduction of Aß40 in neuronally differentiated, patient-derived induced pluripotent stem cells. AON-treated wild-type mice showed successful exon skipping on RNA and protein levels throughout the brain. These results illustrate APP splice modulation as a promising therapeutic approach for D-CAA.


Subject(s)
Amyloid beta-Protein Precursor , Cerebral Amyloid Angiopathy , Amyloid beta-Peptides/genetics , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Brain/metabolism , Cerebral Amyloid Angiopathy/genetics , Cerebral Amyloid Angiopathy/therapy , Humans , Mice , Oligonucleotides, Antisense/genetics
5.
Stem Cell Res ; 39: 101498, 2019 08.
Article in English | MEDLINE | ID: mdl-31326748

ABSTRACT

Huntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an expanded polyglutamine stretch that forms typical protein aggregates throughout the brain. We generated human induced pluripotent stem cells (hiPSCs) from two HD patients using non-integrating Sendai virus (SeV). The hiPSCs display a normal karyotype, express all pluripotency markers, have the same CAG repeat expansion as the original fibroblasts and are able to differentiate into the three germ layers in vitro.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Cells, Cultured , Female , Humans , Huntington Disease/metabolism , Induced Pluripotent Stem Cells/metabolism , Karyotype , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Sendai virus/genetics
6.
Brain Pathol ; 29(6): 793-802, 2019 11.
Article in English | MEDLINE | ID: mdl-30868685

ABSTRACT

In severe forms of cerebral amyloid angiopathy (CAA) pathology, vascular calcification has been observed in the cerebral cortex, both in vivo on MRI and CT, and post-mortem using histopathology. However, the pathomechanisms leading to calcification of CAA-laden arteries are unknown. Therefore, we investigated the correlation between calcification of cortical arterioles and several potential modulators of vascular calcification using immunohistochemistry in a unique collection of brain material of patients with a hereditary form of CAA, namely hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D or D-CAA). We show a topographical association of osteopontin (OPN) and TGFß signaling factor phospho-SMAD2/3 (pSMAD2/3) in calcified CAA vessel walls. OPN and pSMAD2/3 gradually accumulate in vessels prior to calcification. Moreover, we found that the vascular accumulation of Collagen 1 (Col1), OPN and pSMAD2/3 immunomarkers correlated with the CAA severity. This was independently of the vessel size, including capillaries in the most severe cases. We propose that calcification of CAA vessels in the observed HCHWA-D cases may be induced by extracellular OPN trapped in the fibrotic Col1 vessel wall, independently of the presence of vascular amyloid.


Subject(s)
Calcinosis/pathology , Cerebral Amyloid Angiopathy/pathology , Osteopontin/metabolism , Aged , Alzheimer Disease/pathology , Amyloid , Amyloid beta-Peptides/metabolism , Amyloidosis/pathology , Arterioles/pathology , Brain/pathology , Capillaries/metabolism , Cerebral Amyloid Angiopathy/metabolism , Cerebral Amyloid Angiopathy, Familial/pathology , Cerebral Cortex/pathology , Collagen Type I/metabolism , Female , Humans , Male , Middle Aged , Osteopontin/physiology , Phosphorylation , Smad2 Protein/metabolism , Smad3 Protein/metabolism
7.
Stem Cell Res ; 34: 101359, 2019 01.
Article in English | MEDLINE | ID: mdl-30611017

ABSTRACT

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.


Subject(s)
Cell Culture Techniques/methods , Cerebral Amyloid Angiopathy, Familial/pathology , Induced Pluripotent Stem Cells/pathology , Base Sequence , Cell Line , Female , Humans , Middle Aged
8.
Stroke ; 49(9): 2081-2087, 2018 09.
Article in English | MEDLINE | ID: mdl-30354978

ABSTRACT

Background and Purpose- Previous studies of symptomatic and asymptomatic hereditary cerebral amyloid angiopathy (CAA) patients offered the possibility to study the radiological manifestations of CAA in the early stages of the disease. Recently, a striped cortex, observable as hypointense lines perpendicular to the pial surface on T2*-weighted 7T magnetic resonance imaging (MRI), was detected in 40% of the symptomatic hereditary CAA patients. However, the origin of these MRI contrast changes is unknown. This study aimed at defining the underlying pathology associated with the in vivo observed striped pattern. Methods- Formalin-fixed postmortem brain material including the occipital lobe of 4 hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) cases and 6 sporadic CAA cases were selected from local neuropathology tissue collections. Depending on the availability of the material, intact hemispheres or brain slabs including the occipital lobe of these patients were screened for the presence of a striped cortex. Regions containing the striped cortex were then subjected to high-resolution 7T MRI and histopathologic examination. Results- We found 2 hereditary cerebral hemorrhage with amyloidosis-Dutch type cases and 1 sporadic CAA case with striped patterns in the occipital cortex resembling the in vivo signal. Histopathologic examination showed that the striped pattern in the cortex at 7T MRI is because of iron accumulation and calcification of penetrating arteries. The presence of both nonheme iron and calcification on penetrating arteries causes signal loss and hence the abnormal striped patterns in the cortical ribbon on T2*-weighted MRI. Conclusions- We identified iron accumulation and calcification of the vessel wall in hereditary cerebral hemorrhage with amyloidosis-Dutch type as the histopathologic correlates of the striped cortex observed on in vivo 7T MRI.


Subject(s)
Cerebral Amyloid Angiopathy, Familial/diagnostic imaging , Iron/metabolism , Occipital Lobe/diagnostic imaging , Vascular Calcification/diagnostic imaging , Aged , Aged, 80 and over , Cerebral Amyloid Angiopathy/diagnostic imaging , Cerebral Amyloid Angiopathy/metabolism , Cerebral Amyloid Angiopathy/pathology , Cerebral Amyloid Angiopathy, Familial/metabolism , Cerebral Amyloid Angiopathy, Familial/pathology , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Occipital Lobe/metabolism , Occipital Lobe/pathology , Vascular Calcification/pathology
9.
J Alzheimers Dis ; 65(4): 1125-1137, 2018.
Article in English | MEDLINE | ID: mdl-30103327

ABSTRACT

The value of iron-based MRI changes for the diagnosis and staging of Alzheimer's disease (AD) depends on an association between cortical iron accumulation and AD pathology. Therefore, this study determined the cortical distribution pattern of MRI contrast changes in cortical regions selected based on the known distribution pattern of tau pathology and investigated whether MRI contrast changes reflect the underlying AD pathology in the different lobes. T2*-weighted MRI was performed on postmortem cortical tissue of controls, late-onset AD (LOAD), and early-onset AD (EOAD) followed by histology and correlation analyses. Combining ex vivo high-resolution MRI and histopathology revealed that: 1) LOAD and EOAD have a different distribution pattern of AD pathological hallmarks and MRI contrast changes over the cortex, with EOAD showing more severe MRI changes; 2) per lobe, severity of AD pathological hallmarks correlates with iron accumulation, and hence with MRI. Therefore, iron-sensitive MRI sequences allow detection of the cortical distribution pattern of AD pathology ex vivo.


Subject(s)
Alzheimer Disease/pathology , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/metabolism , Image Processing, Computer-Assisted , Iron/metabolism , Magnetic Resonance Imaging , Age of Onset , Aged , Aged, 80 and over , Autopsy , Cerebral Cortex/pathology , Female , Humans , Male , Middle Aged
10.
Front Aging Neurosci ; 10: 102, 2018.
Article in English | MEDLINE | ID: mdl-29706885

ABSTRACT

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) caused by a point mutation resulting in an amino acid change (NP_000475.1:p.Glu693Gln) in the amyloid precursor protein (APP). Post-mortem frontal and occipital cortical brain tissue from nine patients and nine age-related controls was used for RNA sequencing to identify biological pathways affected in HCHWA-D. Although previous studies indicated that pathology is more severe in the occipital lobe in HCHWA-D compared to the frontal lobe, the current study showed similar changes in gene expression in frontal and occipital cortex and the two brain regions were pooled for further analysis. Significantly altered pathways were analyzed using gene set enrichment analysis (GSEA) on 2036 significantly differentially expressed genes. Main pathways over-represented by down-regulated genes were related to cellular aerobic respiration (including ATP synthesis and carbon metabolism) indicating a mitochondrial dysfunction. Principal up-regulated pathways were extracellular matrix (ECM)-receptor interaction and ECM proteoglycans in relation with an increase in the transforming growth factor beta (TGFß) signaling pathway. Comparison with the publicly available dataset from pre-symptomatic APP-E693Q transgenic mice identified overlap for the ECM-receptor interaction pathway, indicating that ECM modification is an early disease specific pathomechanism.

11.
Stem Cell Res ; 29: 125-128, 2018 05.
Article in English | MEDLINE | ID: mdl-29656178

ABSTRACT

Spinocerebellar ataxia type 1 (SCA1) is a hereditary neurodegenerative disease caused by a CAG repeat expansion in exon 8 of the ATXN1 gene. We generated induced pluripotent stem cells (hiPSCs) from a SCA1 patient and his non-affected sister by using non-integrating Sendai Viruses (SeV). The resulting hiPSCs are SeVfree, express pluripotency markers, display a normal karyotype, retain the mutation (length of the CAG repeat expansion in the ATXN1 gene) and are able to differentiate into the three germ layers in vitro.


Subject(s)
Induced Pluripotent Stem Cells/metabolism , Spinocerebellar Ataxias/metabolism , Adult , Cell Differentiation , Cell Line , Female , Humans , Male , Siblings
12.
Brain Pathol ; 28(4): 495-506, 2018 07.
Article in English | MEDLINE | ID: mdl-28557134

ABSTRACT

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid ß (Aß) peptide. Transforming growth factor ß1 (TGFß1) is a key player in vascular fibrosis and in the formation of angiopathic vessels in transgenic mice. Therefore, we investigated whether the TGFß pathway is involved in HCHWA-D pathogenesis in human postmortem brain tissue from frontal and occipital lobes. Components of the TGFß pathway were analyzed with quantitative RT-PCR. TGFß1 and TGFß Receptor 2 (TGFBR2) gene expression levels were significantly increased in HCHWA-D in comparison to the controls, in both frontal and occipital lobes. TGFß-induced pro-fibrotic target genes were also upregulated. We further assessed pathway activation by detecting phospho-SMAD2/3 (pSMAD2/3), a direct TGFß down-stream signaling mediator, using immunohistochemistry. We found abnormal pSMAD2/3 granular deposits specifically on HCHWA-D angiopathic frontal and occipital vessels. We graded pSMAD2/3 accumulation in angiopathic vessels and found a positive correlation with the CAA load independent of the brain area. We also observed pSMAD2/3 granules in a halo surrounding occipital vessels, which was specific for HCHWA-D. The result of this study indicates an upregulation of TGFß1 in HCHWA-D, as was found previously in AD with CAA pathology. We discuss the possible origins and implications of the TGFß pathway deregulation in the microvasculature in HCHWA-D. These findings identify the TGFß pathway as a potential biomarker of disease progression and a possible target of therapeutic intervention in HCHWA-D.


Subject(s)
Cerebral Amyloid Angiopathy, Familial/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Aged , Aged, 80 and over , Cerebral Amyloid Angiopathy, Familial/pathology , Female , Frontal Lobe/blood supply , Frontal Lobe/metabolism , Frontal Lobe/pathology , Humans , Male , Middle Aged , Occipital Lobe/blood supply , Occipital Lobe/metabolism , Occipital Lobe/pathology , Phosphorylation , Signal Transduction , Up-Regulation
13.
Neurobiol Aging ; 62: 231-242, 2018 02.
Article in English | MEDLINE | ID: mdl-29195086

ABSTRACT

Previous MRI studies reported cortical iron accumulation in early-onset (EOAD) compared to late-onset (LOAD) Alzheimer disease patients. However, the pattern and origin of iron accumulation is poorly understood. This study investigated the histopathological correlates of MRI contrast in both EOAD and LOAD. T2*-weighted MRI was performed on postmortem frontal cortex of controls, EOAD, and LOAD. Images were ordinally scored using predefined criteria followed by histology. Nonlinear histology-MRI registration was used to calculate pixel-wise spatial correlations based on the signal intensity. EOAD and LOAD were distinguishable based on 7T MRI from controls and from each other. Histology-MRI correlation analysis of the pixel intensities showed that the MRI contrast is best explained by increased iron accumulation and changes in cortical myelin, whereas amyloid and tau showed less spatial correspondence with T2*-weighted MRI. Neuropathologically, subtypes of Alzheimer's disease showed different patterns of iron accumulation and cortical myelin changes independent of amyloid and tau that may be detected by high-field susceptibility-based MRI.


Subject(s)
Alzheimer Disease/diagnostic imaging , Alzheimer Disease/pathology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Diffusion Magnetic Resonance Imaging , Iron/metabolism , Myelin Sheath/metabolism , Myelin Sheath/pathology , Adult , Aged , Aged, 80 and over , Amyloid beta-Peptides/metabolism , Autopsy , Disease Susceptibility , Female , Humans , Male , Middle Aged , tau Proteins/metabolism
14.
Nucl Med Biol ; 42(8): 695-702, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25960433

ABSTRACT

INTRODUCTION: Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab')2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. METHODS: To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ((111)In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 µg VHH-pa2H-Fc-DTPA-(111)In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. RESULTS: We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50mg/L growth medium. The fusion protein showed homodimerization - necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA-(111)In and the VHH-Fc-DTPA-(111)In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA-(111)In was not improved compared to non-fused VHH-DTPA-(111)In. CONCLUSION: Using VHH-Fc conjugates increases the blood half-life of the protein. However, purely extending the time window for brain uptake does not increase BBB passage. Nevertheless, VHH-Fc holds promise for therapeutic applications where a sustained systemic circulation of VHH is advantageous.


Subject(s)
Amyloid beta-Protein Precursor/physiology , Blood-Brain Barrier/diagnostic imaging , Brain/diagnostic imaging , Brain/metabolism , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Presenilin-1/physiology , Radiopharmaceuticals/pharmacokinetics , Single-Domain Antibodies/pharmacology , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Animals , Blood-Brain Barrier/metabolism , Disease Models, Animal , Fluorescent Antibody Technique , HEK293 Cells , Humans , Image Processing, Computer-Assisted , Indium Radioisotopes/pharmacokinetics , Isotope Labeling , Metabolic Clearance Rate , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pentetic Acid/analogs & derivatives , Pentetic Acid/chemistry , Positron-Emission Tomography/methods , Single-Domain Antibodies/administration & dosage , Tissue Distribution
15.
EJNMMI Res ; 4: 21, 2014.
Article in English | MEDLINE | ID: mdl-24995150

ABSTRACT

BACKGROUND: Previously, we reported on the unexpected development of distant metastases in the subcutaneous rat pancreas CA20948 tumor model after 4.5 weeks of treatment with RAD001-only or in combination with [(177)Lu-DOTA(0),Tyr(3)]octreotate ((177)Lu-DOTATATE) (Cancer Res. 73:12-8, 2013). Moreover, the combination therapy was less effective compared to (177)Lu-DOTATATE-only. In the current study, we address the following questions: (1) Why was the combination therapy less effective? Is (177)Lu-DOTATATE tumor uptake affected by pretreatment with RAD001? (2) Could sudden cessation of RAD001 therapy cause the development of distant metastases? (3) Is (177)Lu-DOTATATE an effective treatment option for these metastases? METHODS: Lewis rats (HanHsd or SsNHsd substrain with a slight difference in immune response) bearing subcutaneous CA20948 tumors were treated with either 125 or 275 MBq (177)Lu-DOTATATE, RAD001, or their combination. RAD001 was given twice a week for 4.5 or 12 weeks, whereas (177)Lu-DOTATATE was given as a single injection. When combined, RAD001 was started either 3 days prior to or 3 days post administration of (177)Lu-DOTATATE. SPECT/CT was performed to quantify (177)Lu-DOTATATE tumor uptake. Where indicated, primary tumors were surgically removed when tumor size is >6,000 mm(3) to enable monitoring for possible metastasis. If metastases were suspected, an (111)In-DTPA-octreotide SPECT/CT scan was performed. Seven rats with metastases were treated with 400 MBq (177)Lu-DOTATATE. RESULTS: Lu-DOTATATE tumor uptake was not significantly affected by RAD001 pretreatment. The occurrence of metastases after RAD001 treatment was not dose dependent in the dose range tested, nor was it related to the duration of RAD001 treatment. In the experiment in which the LEW/SsNsd substrain was used, only 12.5% of RAD001-treated rats showed complete response (CR), compared to 50% tumor regression in the control group. Re-treatment with a high dose of (177)Lu-DOTATATE resulted in CR in only two out of seven animals. CONCLUSION: Less effective anti-tumor effects after the combination of RAD001 + (177)Lu-DOTATATE could not be explained by reduced (177)Lu-DOTATATE tumor uptake after RAD001. Our current data support RAD001-induced immune suppression as the reason for this observation. No evidence was found that cessation of RAD001 treatment caused development of metastases. Metastases appeared to be less sensitive to (177)Lu-DOTATATE treatment than primary tumors.

16.
Arterioscler Thromb Vasc Biol ; 33(8): 1812-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23744990

ABSTRACT

OBJECTIVE: In search of molecular imaging modalities for specific detection of inflammatory atherosclerotic plaques, we explored the potential of targeting scavenger receptor-AI (SR-AI), which is highly expressed by lesional macrophages and linked to effective internalization machinery. APPROACH AND RESULTS: Ultrasmall superparamagnetic iron oxide particles were conjugated to a peptidic SR-AI ligand (0.371 mol Fe/L and 0.018 mol PP1/L). In vitro incubation of human or murine macrophages with SR-AI-targeted USPIO led to significantly higher iron uptake in vitro than with nontargeted USPIO, as judged by quantitative atomic absorption spectroscopy and Perl's staining. Incremental uptake was strictly mediated by SRs. SR-AI-targeted USPIO displayed accelerated plasma decay and a 3.5-fold increase (P=0.01) in atherosclerotic plaque accumulation on intravenous injection into apolipoprotein E-deficient mice compared with nontargeted USPIO. In addition, atherosclerotic humanized LDLr(-/-) chimeras with leukocyte expression of human SR-AI showed a significant improvement in contrast-to-noise ratio (2.7-fold; P=0.003) in the atherosclerotic aortic arch plaques 24 hours after injection of SR-AI-targeted USPIO compared with chimeras with leukocyte SR-AI deficiency. CONCLUSIONS: Collectively, our data provide several lines of evidence that SR-AI-targeted molecular imaging of USPIO-based contrast agents holds great promise for in situ detection of inflammatory plaques in manifest atherosclerosis.


Subject(s)
Dextrans , Magnetic Resonance Imaging/methods , Magnetite Nanoparticles , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology , Scavenger Receptors, Class A/metabolism , Animals , Apolipoproteins E/genetics , Cells, Cultured , Dextrans/pharmacokinetics , Disease Models, Animal , Humans , Macrophages/metabolism , Mice , Mice, Knockout , Nanoparticles , Signal Transduction/physiology , Spectrophotometry, Atomic/methods
17.
PLoS One ; 8(3): e57299, 2013.
Article in English | MEDLINE | ID: mdl-23472079

ABSTRACT

BACKGROUND: High-resolution contrast-enhanced imaging of the murine atherosclerotic vessel wall is difficult due to unpredictable flow artifacts, motion of the thin artery wall and problems with flow suppression in the presence of a circulating contrast agent. METHODS AND RESULTS: We applied a 2D-FLASH retrospective-gated CINE MRI method at 9.4T to characterize atherosclerotic plaques and vessel wall distensibility in the aortic arch of aged ApoE(-/-) mice after injection of a contrast agent. The method enabled detection of contrast enhancement in atherosclerotic plaques in the aortic arch after I.V. injection of micelles and iron oxides resulting in reproducible plaque enhancement. Both contrast agents were taken up in the plaque, which was confirmed by histology. Additionally, the retrospective-gated CINE method provided images of the aortic wall throughout the cardiac cycle, from which the vessel wall distensibility could be calculated. Reduction in plaque size by statin treatment resulted in lower contrast enhancement and reduced wall stiffness. CONCLUSIONS: The retrospective-gated CINE MRI provides a robust and simple way to detect and quantify contrast enhancement in atherosclerotic plaques in the aortic wall of ApoE(-/-) mice. From the same scan, plaque-related changes in stiffness of the aortic wall can be determined. In this mouse model, a correlation between vessel wall stiffness and atherosclerotic lesions was found.


Subject(s)
Aorta/pathology , Atherosclerosis/pathology , Contrast Media/pharmacology , Magnetic Resonance Imaging, Cine/methods , Plaque, Atherosclerotic/pathology , Animals , Apolipoproteins E/genetics , Artifacts , Atherosclerosis/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Micelles , Motion , Plaque, Atherosclerotic/metabolism
18.
J Med Chem ; 56(6): 2374-84, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23427837

ABSTRACT

Novel bombesin (BBN) antagonists were synthesized by coupling the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to H-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (JMV594) through linkers of increasing number of (ßAla)x residues (x = 1-3). Labeling with (111)In afforded the respective radiotracers in high purity and high specific activity. Bioconjugate affinity for the gastrin releasing peptide receptor (GRPR) as determined against [(125)I-Tyr(4)]BBN was high (IC50 values in the lower nanomolar range). Radioligands poorly internalized in PC-3 cells at 37 °C. Radiopeptides remained >60% intact 5 min after entering the bloodstream of healthy mice. After injection in SCID mice bearing human PC-3 xenografts all analogues showed high tumor uptake and rapid background clearance via the kidneys into urine. Interestingly, pancreatic uptake, albeit GRPR-specific, declined rapidly with time. (111)In-DOTA-(ßAla)2-JMV594 achieved the highest tumor values among the group (17.0 ± 2.8%ID/g vs. 8-10%ID/g, respectively, at 4 h pi) indicating that the (ßAla)2-linker favors in vivo interaction of radiopeptides with the GRPR.


Subject(s)
Bombesin/chemical synthesis , Bombesin/pharmacology , Indium Radioisotopes , Receptors, Bombesin/antagonists & inhibitors , Receptors, Bombesin/metabolism , Amino Acid Sequence , Animals , Bombesin/analogs & derivatives , Cell Line, Tumor , Chemistry Techniques, Synthetic , Female , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Isotope Labeling , Ligands , Mice , Oligopeptides/chemistry
19.
Cancer Res ; 73(1): 12-8, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23149918

ABSTRACT

Inhibition of mTOR is commonly considered a valid target in cancer treatment, but this assertion does not address effects on the immune microenvironment that may be detrimental to cancer treatment. Here we show how administration of the mTOR inhibitor RAD001 (everolimus) results in the occurrence of distant metastasis in a rat model of pancreatic cancer. RAD001 was administered twice weekly for 4.5 weeks as a single treatment or combined with [(177)Lu-DOTA,Tyr3]octreotate ((177)Lu-DOTATATE), where the latter targets the somatostatin receptor-2. The hypothesized synergistic therapeutic effect of RAD001 combined with (177)Lu-DOTATATE was, however, not observed in our experiments. The combination was shown to be less effective than (177)Lu-DOTATATE alone. Unexpectedly, tumor metastasis was observed in 77% of the subjects treated with RAD001, either alone or as part of the combination treatment. This was a striking effect, because metastasis did not occur in control or (177)Lu-DOTATATE-treated animals, including those where the primary tumor was surgically removed. These findings may be important clinically among noncompliant patients or patients that discontinue RAD001 therapy because of adverse effects.


Subject(s)
Antineoplastic Agents/adverse effects , Carcinoma, Neuroendocrine/pathology , Pancreatic Neoplasms/pathology , Sirolimus/analogs & derivatives , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Carcinoma, Neuroendocrine/drug therapy , Carcinoma, Neuroendocrine/immunology , Cell Line, Tumor , Disease Models, Animal , Everolimus , Humans , Male , Mice , Mice, Nude , Neoplasm Metastasis/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/immunology , Rats , Rats, Inbred Lew , Sirolimus/adverse effects , TOR Serine-Threonine Kinases/immunology
20.
Contrast Media Mol Imaging ; 8(1): 63-71, 2013.
Article in English | MEDLINE | ID: mdl-23109394

ABSTRACT

The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium-based contrast agents to enhance atherosclerotic plaques in ApoE(-/-) mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer-term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T(1)-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR(664) incorporated in the contrast agents and quantification of tissue and blood Gd-DTPA. Both micelles and liposomes enhanced contrast in T(1)-weighted MR images of plaques in the aortic arch. The average contrast-to-noise ratio increased after liposome or micelle injection to 260 or 280% respectively, at 24 h after injection, compared with a pre-scan. A second wave of maximum contrast enhancement was observed around 60-72 h after injection, which only slowly decreased towards the 1 week end-point. Confocal fluorescence microscopy and whole body fluorescence imaging confirmed MRI-findings of accumulation of micelles and liposomes. Plaque permeation of contrast agents was not strongly dependent on the contrast agent size in this mouse model. Our results show that intraplaque accumulation over time of both contrast agents leads to good plaque visualization for a long period. This inherent intraplaque accumulation might make it difficult to discriminate passive from targeted accumulation. This implies that, in the development of targeted contrast agents on a lipid-based backbone, extensive timing studies are required.


Subject(s)
Aorta, Thoracic/diagnostic imaging , Contrast Media/pharmacology , Gadolinium DTPA/pharmacology , Lipids/pharmacology , Magnetic Resonance Angiography/methods , Micelles , Animals , Aorta, Thoracic/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Contrast Media/pharmacokinetics , Gadolinium DTPA/pharmacokinetics , Kinetics , Lipids/pharmacokinetics , Liposomes/pharmacokinetics , Liposomes/pharmacology , Male , Mice , Mice, Knockout , Plaque, Atherosclerotic , Radiography , Time Factors , Tomography, Optical/methods
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