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Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.
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Envejecimiento , Reparación del ADN , Endonucleasas , Matriz Extracelular , Músculo Liso Vascular , Fenotipo , Animales , Ratones , Envejecimiento/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/deficiencia , Endonucleasas/metabolismo , Endonucleasas/deficiencia , Endonucleasas/genética , Matriz Extracelular/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismoRESUMEN
Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85-0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases.
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INTRODUCTION: Early detection of liver fibrosis and monitoring response to treatment crucial for the management of patients are currently not feasible in clinical practice. Platelet derived growth factor receptor ß (PDGFR-ß) expression is regarded as a potential biomarker to determine the stages of fibrotic diseases including liver fibrosis. [68Ga]Ga-BOT5035 comprising a bicyclic peptide was developed for specific targeting of PDGFR-ß overexpressed in pathological fibrosis. The realization of microdosing phase 0 study using [68Ga]Ga-BOT5035 positron emission tomography required automated good manufacturing practice (GMP) compliant production of [68Ga]Ga-BOT5035 presented herein. Moreover, the investigation of radiation dosimetry was conducted to ensure possibility of multiple annual examinations for disease monitoring in clinical setup. METHODS: The active pharmaceutical ingredient starting material BOT5035 (GMP grade) was provided by BiOrion Technologies BV. The 68Ga-labelling process was developed and automated using synthesis platform (Modular-Lab PharmTrace, Eckert & Ziegler), disposable cassettes for 68Ga-labelling, and pharmaceutical grade 68Ge/68Ga generator (GalliaPharm®) purchased from Eckert & Ziegler. Radiolysis sensitive BOT5035 required development and systematic optimization of the labelling synthesis parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration and pH. The validation process was conducted with regard to the product quality and quantity, as well as production reproducibility. Human organ equivalent doses and total body effective doses were calculated using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1), based on ex vivo organ distribution in Sprague-Dawley rats. RESULTS: The GMP compliant automated production of [68Ga]Ga-BOT5035 with on-line documentation demonstrated high reproducibility. The time for the labelling synthesis and quality control was approximately 60â¯min. The non-decay corrected radiochemical yield and radiochemical purity of the radiopharmaceutical were 43.7⯱â¯7.6% (nâ¯=â¯3, process validation) and 97.7⯱â¯0.4% (nâ¯=â¯3, process validation), respectively. Predefined acceptance criteria were met for the sterility, endotoxins level, radionuclidic purity and residual solvent content. The stability at ambient temperature was controlled for 120â¯min with approved results. Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. The dose-limiting organs were kidney and bone marrow. The total effective dose as limiting parameter would allow for up to 3-4 PET scans per annum. CONCLUSION: The fully automated and GMP compliant production of [68Ga]Ga-BOT5035 was developed and thoroughly validated. The radiopharmaceutical was approved by Swedish Medicinal Products Agency and the Ethical Review Authority for the Phase 0 clinical study of the quantitative imaging of liver fibrosis. Human dosimetry calculations extrapolated from animal experiment indicated possibility of 3-4 PET examinations per year.
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Industria Farmacéutica/normas , Radioisótopos de Galio/metabolismo , Cirrosis Hepática/patología , Péptidos Cíclicos/metabolismo , Radiofármacos/metabolismo , Animales , Ensayos Clínicos como Asunto , Femenino , Humanos , Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The heat shock protein (HSP) inducer, geranylgeranylacetone (GGA), was previously found to protect against atrial fibrillation (AF) remodeling in experimental model systems. Clinical application of GGA in AF is limited, due to low systemic concentrations owing to the hydrophobic character of GGA. OBJECTIVES: To identify novel HSP-inducing compounds, with improved physicochemical properties, that prevent contractile dysfunction in experimental model systems for AF. METHODS: Eighty-one GGA-derivatives were synthesized and explored for their HSP-inducing properties by assessment of HSP expression in HL-1 cardiomyocytes pretreated with or without a mild heat shock (HS), followed by incubation with 10 µM GGA or GGA-derivative. Subsequently, the most potent HSP-inducers were tested for preservation of calcium transient (CaT) amplitudes or heart wall contraction in pretreated tachypaced HL-1 cardiomyocytes (with or without HSPB1 siRNA) and Drosophilas, respectively. Finally, CaT recovery in tachypaced HL-1 cardiomyocytes posttreated with GGA or protective GGA-derivatives was determined. RESULTS: Thirty GGA-derivatives significantly induced HSPA1A expression after HS, and seven showed exceeding HSPA1A expression compared to GGA. GGA and nine GGA-derivatives protected significantly from tachypacing (TP)-induced CaT loss, which was abrogated by HSPB1 suppression. GGA and four potent GGA-derivatives protected against heart wall dysfunction after TP compared to non-paced control Drosophilas. Of these compounds, GGA and three GGA-derivatives induced a significant restoration from CaT loss after TP of HL-1 cardiomyocytes. CONCLUSION: We identified novel GGA-derivatives with improved physicochemical properties compared to GGA. GGA-derivatives, particularly GGA*-59, boost HSP expression resulting in prevention and restoration from TP-induced remodeling, substantiating their role as novel therapeutics in clinical AF.
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Fibrilación Atrial/tratamiento farmacológico , Diterpenos/farmacología , Proteínas de Choque Térmico/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Animales , Fibrilación Atrial/metabolismo , Diterpenos/síntesis química , Diterpenos/química , Relación Dosis-Respuesta a Droga , Drosophila , Evaluación Preclínica de Medicamentos , Estructura Molecular , Miocitos Cardíacos/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Atrial fibrillation is a progressive arrhythmia, the exact mechanism underlying the progressive nature of recurrent AF episodes is still unknown. Recently, it was found that key players of the protein quality control system of the cardiomyocyte, i.e. Heat Shock Proteins, protect against atrial fibrillation progression by attenuating atrial electrical and structural remodeling (electropathology). HALT & REVERSE aims to investigate the correlation between electropathology, as defined by endo- or epicardial mapping, Heat Shock Protein levels and development or recurrence of atrial fibrillation following pulmonary vein isolation, or electrical cardioversion or cardiothoracic surgery. STUDY DESIGN: This study is a prospective observational study. Three separate study groups are defined: (1) cardiothoracic surgery, (2) pulmonary vein isolation and (3) electrical cardioversion. An intra-operative high-resolution epicardial (group 1) or endocardial (group 2) mapping procedure of the atria is performed to study atrial electropathology. Blood samples for Heat Shock Protein determination are obtained at baseline and during the follow-up period at 3 months (group 2), 6 months (groups 1 and 2) and 1 year (group 1 and 2). Tissue samples of the right and left atrial appendages in patients in group 1 are analysed for Heat Shock Protein levels and for tissue characteristics. Early post procedural atrial fibrillation is detected by continuous rhythm monitoring, whereas late post procedural atrial fibrillation is documented by either electrocardiogram or 24-h Holter registration. CONCLUSION: HALT & REVERSE aims to identify the correlation between Heat Shock Protein levels and degree of electropathology. The study outcome will contribute to novel diagnostic tools for the early recognition of clinical atrial fibrillation. TRIAL REGISTRATIONS: Rotterdam Medical Ethical Committee MEC-2014-393, Dutch Trial Registration NTR4658.
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Fibrilación Atrial/prevención & control , Fibrilación Atrial/terapia , Proteínas de Unión al ADN/sangre , Miocitos Cardíacos/metabolismo , Factores de Transcripción/sangre , Adolescente , Adulto , Anciano , Fibrilación Atrial/cirugía , Puente Cardiopulmonar , Electrocardiografía , Femenino , Atrios Cardíacos/patología , Factores de Transcripción del Choque Térmico , Humanos , Periodo Intraoperatorio , Masculino , Persona de Mediana Edad , Pericardio/patología , Estudios Prospectivos , Venas Pulmonares/fisiopatología , Recurrencia , Proyectos de Investigación , Resultado del Tratamiento , Adulto JovenRESUMEN
Tumor-targeting of anticancer drugs is an interesting approach for the treatment of cancer since chemotherapies possess several adverse effects. In the present study, we propose a novel strategy to deliver anticancer drugs to the tumor cells through the mannose-6-phosphate/insulin-like growth factor receptor (M6P/IGF-IIR) which are abundantly expressed in several human tumors. We developed a drug carrier against M6P/IGF-II receptor by modifying human serum albumin (HSA) with M6P moieties. M6P-HSA specifically bound and internalized into M6P/IGF-IIR-expressing B16 melanoma cells as demonstrated with radioactive studies and anti-HSA immunostaining. In vivo, M6P-HSA rapidly accumulated in subcutaneous tumors in tumor and stromal components after an intravenous injection. To demonstrate the application of M6P-HSA as a drug carrier, we coupled doxorubicin to it. Dox-HSA-M6P conjugate could release doxorubicin at lysosomal pH and showed M6P-specific binding and uptake in tumor cells. In vitro, a short exposure with Dox-HSA-M6P induced killing of tumor cells, which could be blocked by excess M6P-HSA. In vivo, Dox-HSA-M6P distributed to tumors and some other organs while free doxorubicin distributed to all organs but slightly to tumors. In B16 tumor-bearing mice, Dox-HSA-M6P significantly inhibited the tumor growth whereas an equimolar dose of free doxorubicin did not show any anti-tumor effect. In addition, targeted doxorubicin did not show any side-effects on liver and kidney function tests, body weight and blood cell counts. In conclusion, M6P-HSA is a suitable carrier for delivery of anticancer drugs to tumors through M6P/IGF-IIR. Improved antitumor effects of the targeted doxorubicin by M6P-HSA suggest that this novel approach may be applied to improve the therapeutic efficacy of anticancer drugs.