Adult mammalian hearts cannot repair by themselves after injury due to limited proliferation of cardiomyocytes; removal of cell cycle blocker and/or addition of drugs that boost proliferation of ...cardiomyocytes provide potential means to cardiac regeneration. Three publications that appeared recently in Nature and Cell Research now provide new hope to the treatment of heart injuries.
•An exploratory randomized, controlled trial of baloxavir marboxil and favipiravir in COVID-19 patients were conducted.•The free drug concentrations of baloxavir acid and favipiravir are generally ...lower than their respective EC50 values.•Add-on either baloxavir or favipiravir to the current standard treatment resulted in no additional antiviral benefit.
Background: Effective antiviral drugs for COVID-19 are still lacking. This study aims to evaluate the clinical outcomes and plasma concentrations of baloxavir acid and favipiravir in COVID-19 patients.
Methods: Favipiravir and baloxavir acid were evaluated for their antiviral activity against SARS-CoV-2 in vitro before the trial initiation. We conducted an exploratory trial with 3 arms involving hospitalized adult patients with COVID-19. Patients were randomized assigned in a 1:1:1 ratio into baloxavir marboxil group, favipiravir group, and control group. The primary outcome was the percentage of subjects with viral negative by Day 14 and the time from randomization to clinical improvement. Virus load reduction, blood drug concentration and clinical presentation were also observed. The trial was registered with Chinese Clinical Trial Registry (ChiCTR 2000029544).
Results: Baloxavir acid showed antiviral activity in vitro with the half-maximal effective concentration (EC50) of 5.48 μM comparable to arbidol and lopinavir, but favipiravir didn't demonstrate significant antiviral activity up to 100 μM. Thirty patients were enrolled. The percentage of patients who turned viral negative after 14-day treatment was 70%, 77%, and 100% in the baloxavir marboxil, favipiravir, and control group respectively, with the medians of time from randomization to clinical improvement was 14, 14 and 15 days, respectively. One reason for the lack of virological effect and clinical benefits may be due to insufficient concentrations of these drugs relative to their antiviral activities. One of the limitations of this study is the time from symptom onset to randomization, especially in the baloxavir marboxil and control groups, which is higher than the favipiravir group.
Conclusions: Our findings could not prove a benefit of addition of either baloxavir marboxil or favipiravir under the trial dosages to the existing standard treatment.
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Organ homeostasis is orchestrated by time- and spatially restricted cell proliferation. Studies identifying cells with superior proliferative capacities often rely on the lineage tracing of a subset ...of cell populations, which introduces a potential selective bias. In this work, we developed a genetic system proliferation tracer (ProTracer) by incorporating dual recombinases to seamlessly record the proliferation events of entire cell populations over time in multiple organs. In the mouse liver, ProTracer revealed more hepatocyte proliferation in distinct zones during liver homeostasis, injury repair, and regrowth. Clonal analysis showed that most of the hepatocytes labeled by ProTracer had undergone cell division. By genetically recording proliferation events of entire cell populations, ProTracer enables the unbiased detection of specific cellular compartments with enhanced regenerative capacities.
Abstract
Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information ...to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration.
Organs of the body require vascular networks to supply oxygen and nutrients and maintain physiological function. The blood vessels of different organs are structurally and functionally heterogeneous ...in nature. To more precisely dissect their distinct in vivo function in individual organs, without potential interference from off-site targets, it is necessary to genetically target them in an organ-specific manner.
The objective of this study was to generate a genetic system that targets vascular endothelial cells in an organ- or tissue-specific manner and to exemplify the potential application of intersectional genetics for precise, target-specific gene manipulation in vivo.
We took advantage of 2 orthogonal recombination systems, Dre-rox and Cre-loxP, to create a genetic targeting system based on intersectional genetics. Using this approach, Cre activity was only detectable in cells that had expressed both Dre and Cre. Applying this new system, we generated a coronary endothelial cell-specific Cre (
) and a brain endothelial cell-specific Cre (
). Through lineage tracing, gene knockout and overexpression experiments, we demonstrated that
and
efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of vascular endothelial growth factor receptor 2 using
, we showed that vascular endothelial growth factor signaling regulates angiogenesis in the central nervous system and also controls the integrity of the blood-brain barrier.
We provide 2 examples to illustrate the use of intersectional genetics for more precise gene targeting in vivo, namely manipulation of genes in blood vessels of the heart and brain. More broadly, this system provides a valuable strategy for tissue-specific gene manipulation that can be widely applied to other fields of biomedical research.
The Cre-loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity ...of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre-rox recombination system to enhance the precision of conventional Cre-loxP-mediated lineage tracing. The Dre-rox system permits rigorous control of Cre-loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates-the contribution of c-Kit
cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9
hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre-loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.
The use of the dual recombinase-mediated intersectional genetic approach involving Cre-loxP and Dre-rox has significantly enhanced the precision of in vivo lineage tracing, as well as gene ...manipulation. However, this approach is limited by the small number of Dre recombinase driver constructs available. Here, we developed more than 70 new intersectional drivers to better target diverse cell lineages. To highlight their applicability, we used these new tools to study the in vivo adipogenic fate of perivascular progenitors, which revealed that PDGFRa+ but not PDGFRa–PDGFRb+ perivascular cells are the endogenous progenitors of adult adipocytes. In addition to lineage tracing, we used members of this new suite of drivers to more specifically knock out genes in complex tissues, such as white adipocytes and lymphatic vessels, that heretofore cannot be selectively targeted by conventional Cre drivers alone. In summary, these new transgenic tools expand the intersectional genetic approach while enhancing its precision.
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•More than 70 new Dre driver lines are provided as a resource for intersectional genetics•PDGFRa+PDGFRb+ or PDGFRa+ perivascular cells contribute to de novo adipocytes•“Exclusion” dual recombinase enables gene deletion in white but not brown adipocytes•Sequential dual recombinase enables gene deletion in lymphatic endothelial cells
The combinatory use of Dre and Cre recombinase-mediated intersectional genetics significantly enhances the precision of in vivo lineage tracing and gene targeting. Han et al. developed more than 70 new intersectional drivers to target diverse cell lineages. Highlighting their application, Han et al. used these new tools to study perivascular progenitors of adipocytes and performed gene knockout in white adipocytes (WAs) and lymphatic endothelial cells (LECs).
Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple ...sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus （SV）, and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells （ECs） both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepieardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.
During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum ...and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages.
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