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1.
Arterioscler Thromb Vasc Biol ; 42(4): 381-394, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35172604

RESUMEN

BACKGROUND: The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. METHODS: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. RESULTS: Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. CONCLUSIONS: Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.


Asunto(s)
Intestinos , Células Madre , Acilcoenzima A , Animales , Colesterol , Femenino , Masculino , Ratones , Esteroles
2.
Eur Radiol ; 32(4): 2629-2638, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34812912

RESUMEN

OBJECTIVE: To systematically review and evaluate the methodological quality of studies using magnetic resonance imaging (MRI) and computed tomography (CT) radiomics for cardiac applications. METHODS: Multiple medical literature archives (PubMed, Web of Science, and EMBASE) were systematically searched to retrieve original studies focused on cardiac MRI and CT radiomics applications. Two researchers in consensus assessed each investigation using the radiomics quality score (RQS). Subgroup analyses were performed to assess whether the total RQS varied according to study aim, journal quartile, imaging modality, and first author category. RESULTS: From a total of 1961 items, 53 articles were finally included in the analysis. Overall, the studies reached a median total RQS of 7 (IQR, 4-12), corresponding to a percentage score of 19.4% (IQR, 11.1-33.3%). Item scores were particularly low due to lack of prospective design, cost-effectiveness analysis, and open science. Median RQS percentage score was significantly higher in papers where the first author was a medical doctor and in those published on first quartile journals. CONCLUSIONS: The overall methodological quality of radiomics studies in cardiac MRI and CT is still lacking. A higher degree of standardization of the radiomics workflow and higher publication standards for studies are required to allow its translation into clinical practice. KEY POINTS: • RQS has been recently proposed for the overall assessment of the methodological quality of radiomics-based studies. • The 53 included studies on cardiac MRI and CT radiomics applications reached a median total RQS of 7 (IQR, 4-12), corresponding to a percentage of 19.4% (IQR, 11.1-33.3%). • A more standardized methodology in the radiomics workflow is needed, especially in terms of study design, validation, and open science, in order to translate the results to clinical applications.


Asunto(s)
Imagen por Resonancia Magnética , Tomografía Computarizada por Rayos X , Humanos , Imagen por Resonancia Magnética/métodos , Radiografía
3.
J Cardiovasc Magn Reson ; 24(1): 31, 2022 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-35606874

RESUMEN

BACKGROUND: T1 mapping is an established cardiovascular magnetic resonance (CMR) technique that can characterize myocardial tissue. We aimed to determine the weighted mean native T1 values of Anderson-Fabry disease (AFD) patients and the standardized mean differences (SMD) as compared to healthy control subjects. METHODS: A comprehensive literature search of the PubMed, Scopus and Web of Science databases was conducted according to the PRISMA statement to retrieve original studies reporting myocardial native T1 values in AFD patients and healthy controls. A random effects model was used to calculate SMD, and meta-regression analysis was conducted to explore heterogeneity sources. Subgroup analysis was also performed according to scanner field strength and sequence type. RESULTS: From a total of 151 items, 14 articles were included in the final analysis accounting for a total population of 982 subjects. Overall, the weighted mean native T1 values was 984 ± 47 ms in AFD patients and 1016 ± 26 ms in controls (P < 0.0001) with a pooled SMD of - 2.38. In AFD patients there was an inverse correlation between native T1 values and male gender (P = 0.002) and left ventricular hypertrophy (LVH) (P < 0.001). Subgroup analyses confirmed lower T1 values in AFD patients compared to controls with a pooled SMD of -  2.54, -  2.28, -  2.46 for studies performed on 1.5T with modified Look-Locker inversion recovery (MOLLI), shortened MOLLI and saturation-recovery single-shot acquisition, respectively and of -  2.41 for studies conducted on 3T. CONCLUSIONS: Our findings confirm a reduction of native T1 values in AFD patients compared to healthy controls and point out that the degree of T1 shortening in AFD is influenced by gender and LVH. Although T1 mapping is useful in proving cardiac involvement in AFD patients, there is need to standardize shreshold values according to imaging equipment and protocols.


Asunto(s)
Enfermedad de Fabry , Corazón , Humanos , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/etiología , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética , Masculino , Valor Predictivo de las Pruebas
4.
Mol Ther ; 28(6): 1432-1441, 2020 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-32348718

RESUMEN

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8+ T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.


Asunto(s)
Proteína 9 Asociada a CRISPR/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dependovirus/genética , Edición Génica/métodos , Vectores Genéticos/genética , Animales , Biomarcadores , Proteína 9 Asociada a CRISPR/efectos adversos , Expresión Génica , Orden Génico , Hepatocitos/metabolismo , Humanos , Inmunización , Memoria Inmunológica , Inmunofenotipificación , Ratones , ARN Guía de Kinetoplastida , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transgenes
5.
J Lipid Res ; 61(12): 1675-1686, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33109681

RESUMEN

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.


Asunto(s)
Estrés del Retículo Endoplásmico/genética , Hidroximetilglutaril-CoA Reductasas/deficiencia , Hidroximetilglutaril-CoA Reductasas/genética , Hígado/metabolismo , Terpenos/metabolismo , Eliminación de Gen
6.
Arterioscler Thromb Vasc Biol ; 38(9): 1997-2006, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30026278

RESUMEN

Objective- Atherosclerosis studies in Ldlr knockout mice require breeding to homozygosity and congenic status on C57BL6/J background, a process that is both time and resource intensive. We aimed to develop a new method for generating atherosclerosis through somatic deletion of Ldlr in livers of adult mice. Approach and Results- Overexpression of PCSK9 (proprotein convertase subtilisin/kexin type 9) is currently used to study atherosclerosis, which promotes degradation of LDLR (low-density lipoprotein receptor) in the liver. We sought to determine whether CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) could also be used to generate atherosclerosis through genetic disruption of Ldlr in adult mice. We engineered adeno-associated viral (AAV) vectors expressing Staphylococcus aureus Cas9 and a guide RNA targeting the Ldlr gene (AAV-CRISPR). Both male and female mice received either (1) saline, (2) AAV-CRISPR, or (3) AAV-hPCSK9 (human PCSK9)-D374Y. A fourth group of germline Ldlr-KO mice was included for comparison. Mice were placed on a Western diet and followed for 20 weeks to assess plasma lipids, PCSK9 protein levels, atherosclerosis, and editing efficiency. Disruption of Ldlr with AAV-CRISPR was robust, resulting in severe hypercholesterolemia and atherosclerotic lesions in the aorta. AAV-hPCSK9 also produced hypercholesterolemia and atherosclerosis as expected. Notable sexual dimorphism was observed, wherein AAV-CRISPR was superior for Ldlr removal in male mice, while AAV-hPCSK9 was more effective in female mice. Conclusions- This all-in-one AAV-CRISPR vector targeting Ldlr is an effective and versatile tool to model atherosclerosis with a single injection and provides a useful alternative to the use of germline Ldlr-KO mice.


Asunto(s)
Aterosclerosis/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Modelos Animales de Enfermedad , Vectores Genéticos , Receptores de LDL/genética , Adenoviridae , Animales , Aterosclerosis/sangre , Proteína 9 Asociada a CRISPR/genética , Femenino , Edición Génica , Expresión Génica , Hipercolesterolemia/sangre , Hipercolesterolemia/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/genética , Receptores de LDL/sangre
7.
J Nucl Cardiol ; 26(3): 857-865, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-29076052

RESUMEN

BACKGROUND: To compare cardiac magnetic resonance (CMR) qualitative and quantitative analysis methods for the noninvasive assessment of myocardial inflammation in patients with suspected acute myocarditis (AM). METHODS: A total of 61 patients with suspected AM underwent coronary angiography and CMR. Qualitative analysis was performed applying Lake-Louise Criteria (LLC), followed by quantitative analysis based on the evaluation of edema ratio (ER) and global relative enhancement (RE). Diagnostic performance was assessed for each method by measuring the area under the curves (AUC) of the receiver operating characteristic analyses. The final diagnosis of AM was based on symptoms and signs suggestive of cardiac disease, evidence of myocardial injury as defined by electrocardiogram changes, elevated troponin I, exclusion of coronary artery disease by coronary angiography, and clinical and echocardiographic follow-up at 3 months after admission to the chest pain unit. RESULTS: In all patients, coronary angiography did not show significant coronary artery stenosis. Troponin I levels and creatine kinase were higher in patients with AM compared to those without (both P < .001). There were no significant differences among LLC, T2-weighted short inversion time inversion recovery (STIR) sequences, early (EGE), and late (LGE) gadolinium-enhancement sequences for diagnosis of AM. The AUC for qualitative (T2-weighted STIR 0.92, EGE 0.87 and LGE 0.88) and quantitative (ER 0.89 and global RE 0.80) analyses were also similar. CONCLUSIONS: Qualitative and quantitative CMR analysis methods show similar diagnostic accuracy for the diagnosis of AM. These findings suggest that a simplified approach using a shortened CMR protocol including only T2-weighted STIR sequences might be useful to rule out AM in patients with acute coronary syndrome and normal coronary angiography.


Asunto(s)
Imagen por Resonancia Magnética/métodos , Miocarditis/diagnóstico por imagen , Enfermedad Aguda , Adulto , Estudios de Cohortes , Angiografía Coronaria , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocarditis/complicaciones , Miocarditis/fisiopatología , Valor Predictivo de las Pruebas , Curva ROC , Volumen Sistólico , Evaluación de Síntomas
8.
J Lipid Res ; 58(7): 1292-1305, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28487312

RESUMEN

Cd39 scavenges extracellular ATP and ADP, ultimately generating adenosine, a nucleoside, which has anti-inflammatory effects in the vasculature. We have evaluated the role of Cd39 in the development of atherosclerosis in hyperlipidemic mice. ApoE KO (Cd39+/+/ApoE-/-) and Cd39/ApoE double KO (DKO) (Cd39-/-/ApoE-/-) mice were maintained on chow or Western diet for up to 20 weeks before evaluation of atherosclerotic lesions. We found that DKO mice exhibited significantly fewer atherosclerotic lesions than ApoE KO mice, irrespective of diet. Analyses of plaque composition revealed diminished foam cells in the fatty streaks and smaller necrotic cores in advanced lesions of DKO mice, when compared with those in ApoE KO mice. This atheroprotective phenotype was associated with impaired platelet reactivity to ADP in vitro and prolonged platelet survival, suggesting decreased platelet activation in vivo. Further studies with either genetic deletion or pharmacological inhibition of Cd39 in macrophages revealed increased cholesterol efflux mediated via ABCA1 to ApoA1. This phenomenon was associated with elevated plasma HDL levels in DKO mice. Our findings indicate that complete deletion of Cd39 paradoxically attenuates development of atherosclerosis in hyperlipidemic mice. We propose that this phenotype occurs, at least in part, from diminished platelet activation, increased plasma HDL levels, and enhanced cholesterol efflux and indicates the complexity of purinergic signaling in atherosclerosis.


Asunto(s)
Antígenos CD/genética , Apolipoproteínas E/deficiencia , Apirasa/deficiencia , Apirasa/genética , Aterosclerosis/genética , Técnicas de Inactivación de Genes , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Transporte Biológico/genética , Movimiento Celular/genética , Proliferación Celular/genética , Colesterol/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Músculo Liso Vascular/patología , Necrosis/genética , Fenotipo , Activación Plaquetaria/genética
9.
Plasmid ; 79: 22-9, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25779031

RESUMEN

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.


Asunto(s)
5'-Nucleotidasa/genética , Antígenos CD/genética , Apirasa/genética , Regulación de la Expresión Génica , Hemo-Oxigenasa 1/genética , 5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Furina/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Hemo-Oxigenasa 1/metabolismo , Humanos , Plásmidos/genética , Trasplante Heterólogo
10.
Explor Target Antitumor Ther ; 4(4): 545-555, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37720347

RESUMEN

In the past few years, artificial intelligence (AI) has been increasingly used to create tools that can enhance workflow in medicine. In particular, neuro-oncology has benefited from the use of AI and especially machine learning (ML) and radiogenomics, which are subfields of AI. ML can be used to develop algorithms that dynamically learn from available medical data in order to automatically do specific tasks. On the other hand, radiogenomics can identify relationships between tumor genetics and imaging features, thus possibly giving new insights into the pathophysiology of tumors. Therefore, ML and radiogenomics could help treatment tailoring, which is crucial in personalized neuro-oncology. The aim of this review is to illustrate current and possible future applications of ML and radiomics in neuro-oncology.

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