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1.
Nature ; 595(7865): 107-113, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33915569

RESUMEN

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.


Asunto(s)
COVID-19/patología , COVID-19/virología , Riñón/patología , Hígado/patología , Pulmón/patología , Miocardio/patología , SARS-CoV-2/patogenicidad , Adulto , Anciano , Anciano de 80 o más Años , Atlas como Asunto , Autopsia , Bancos de Muestras Biológicas , COVID-19/genética , COVID-19/inmunología , Células Endoteliales , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Fibroblastos , Estudio de Asociación del Genoma Completo , Corazón/virología , Humanos , Inflamación/patología , Inflamación/virología , Riñón/virología , Hígado/virología , Pulmón/virología , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Fagocitos , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , ARN Viral/análisis , Regeneración , SARS-CoV-2/inmunología , Análisis de la Célula Individual , Carga Viral
2.
J Am Heart Assoc ; 11(1): e022010, 2022 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-34970914

RESUMEN

Background Myocardial injury in patients with COVID-19 is associated with increased mortality during index hospitalization; however, the relationship to long-term sequelae of SARS-CoV-2 is unknown. This study assessed the relationship between myocardial injury (high-sensitivity cardiac troponin T level) during index hospitalization for COVID-19 and longer-term outcomes. Methods and Results This is a prospective cohort of patients who were hospitalized at a single center between March and May 2020 with SARS-CoV-2. Cardiac biomarkers were systematically collected. Outcomes were adjudicated and stratified on the basis of myocardial injury. The study cohort includes 483 patients who had high-sensitivity cardiac troponin T data during their index hospitalization. During index hospitalization, 91 (18.8%) died, 70 (14.4%) had thrombotic complications, and 126 (25.6%) had cardiovascular complications. By 12 months, 107 (22.2%) died. During index hospitalization, 301 (62.3%) had cardiac injury (high-sensitivity cardiac troponin T≧14 ng/L); these patients had 28.6%, 32.2%, and 33.2% mortality during index hospitalization, at 6 months, and at 12 months, respectively, compared with 4.1%, 4.9%, and 4.9% mortality for those with low-level positive troponin and 0%, 0%, and 0% for those with undetectable troponin. Of 392 (81.2%) patients who survived the index hospitalization, 94 (24%) had at least 1 readmission within 12 months, of whom 61 (65%) had myocardial injury during the index hospitalization. Of 377 (96%) patients who were alive and had follow-up after the index hospitalization, 211 (56%) patients had a documented, detailed clinical assessment at 6 months. A total of 78 of 211 (37.0%) had ongoing COVID-19-related symptoms; 34 of 211 (16.1%) had neurocognitive decline, 8 of 211 (3.8%) had increased supplemental oxygen requirements, and 42 of 211 (19.9%) had worsening functional status. Conclusions Myocardial injury during index hospitalization for COVID-19 was associated with increased mortality and may predict who are more likely to have postacute sequelae of COVID-19. Among patients who survived their index hospitalization, the incremental mortality through 12 months was low, even among troponin-positive patients.


Asunto(s)
COVID-19 , Lesiones Cardíacas , COVID-19/complicaciones , COVID-19/terapia , Lesiones Cardíacas/epidemiología , Hospitalización , Humanos , Estudios Prospectivos , Resultado del Tratamiento , Troponina T/sangre
3.
Nat Genet ; 54(12): 1803-1815, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36474045

RESUMEN

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.


Asunto(s)
Enfermedad de la Arteria Coronaria , Humanos , Enfermedad de la Arteria Coronaria/genética , Estudio de Asociación del Genoma Completo
4.
Curr Protoc ; 1(11): e291, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34748284

RESUMEN

Genome editing of primary human cells with CRISPR-Cas9 is a powerful tool to study gene function. For many cell types, there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing due to low transfection efficiency, poor expression of the Cas9 machinery, and toxic effects of the selection antibiotics. Here, we describe a method for high-efficiency editing of primary human vascular cells in vitro using nucleofection for direct delivery of sgRNA:Cas9-NLS ribonucleoprotein complexes. This method is more rapid and its high editing efficiency eliminates the need for additional selection steps. The edited cells can be employed in diverse applications, such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques using viral plasmid delivery. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing. © 2021 Wiley Periodicals LLC. Basic Protocol: CRISPR-Cas9 genome editing of primary human vascular cells in vitro.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Expresión Génica , Humanos , Plásmidos , Transfección
5.
Circ Genom Precis Med ; 14(5): e003399, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34592835

RESUMEN

BACKGROUND: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). METHODS: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. RESULTS: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). CONCLUSIONS: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.


Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Genómica , Mutación Missense , Análisis de Secuencia de ADN , Serina Endopeptidasas/genética , Adulto , Enfermedad de la Arteria Coronaria/epidemiología , Femenino , Humanos , Italia/epidemiología , Masculino , Factores de Riesgo
6.
Blood Adv ; 5(5): 1164-1177, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33635335

RESUMEN

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation.


Asunto(s)
COVID-19/inmunología , Activación Neutrófila , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , COVID-19/sangre , COVID-19/mortalidad , Enfermedad Crítica/epidemiología , Enfermedad Crítica/mortalidad , Estudios Transversales , Femenino , Hospitalización , Humanos , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Pronóstico , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad
7.
bioRxiv ; 2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33655247

RESUMEN

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

8.
medRxiv ; 2020 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-32908988

RESUMEN

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of over 3,300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, HGF, IL-8, and G-CSF, as the strongest predictors of critical illness. Neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, we define an essential role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular neutrophil markers that distinguish patients at risk of future clinical decompensation.

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