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1.
Commun Biol ; 6(1): 370, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-37016073

RESUMO

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.


Assuntos
Melaninas , Melanoma , Camundongos , Animais , Melaninas/metabolismo , Diagnóstico por Imagem , Melanoma/patologia , Encéfalo/metabolismo , Corantes , Mamíferos
2.
Mol Pharm ; 19(10): 3576-3585, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-35434995

RESUMO

Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.


Assuntos
Proteínas de Repetição de Anquirina Projetadas , Receptor ErbB-2 , Animais , Linhagem Celular Tumoral , Desferroxamina/química , Feminino , Humanos , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Receptor ErbB-2/metabolismo , Distribuição Tecidual , Zircônio/química
3.
Cardiovasc Res ; 118(1): 254-266, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33483748

RESUMO

AIMS: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. METHODS AND RESULTS: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. CONCLUSIONS: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.


Assuntos
Aorta Torácica/efeitos dos fármacos , Arginase/metabolismo , Artrite Reumatoide/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Receptores Depuradores Classe E/metabolismo , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Vasodilatação/efeitos dos fármacos , Adulto , Animais , Animais Geneticamente Modificados , Aorta Torácica/enzimologia , Aorta Torácica/imunologia , Aorta Torácica/fisiopatologia , Arginase/genética , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores Depuradores Classe E/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
4.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34001602

RESUMO

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.


Assuntos
Anticorpos/farmacologia , Sistemas de Liberação de Medicamentos , Terapia Genética , Neoplasias/tratamento farmacológico , Adenoviridae/genética , Animais , Anticorpos/genética , Anticorpos/imunologia , Antígenos de Superfície/genética , Antineoplásicos/farmacologia , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Imageamento Tridimensional , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Comunicação Parácrina/efeitos dos fármacos
5.
Atherosclerosis ; 304: 30-38, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32574829

RESUMO

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.


Assuntos
Quimiocina CXCL12 , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Fator A de Crescimento do Endotélio Vascular , Animais , Modelos Animais de Doenças , Células Endoteliais , Membro Posterior , Músculo Esquelético , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Fator A de Crescimento do Endotélio Vascular/farmacologia
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