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.
BACKGROUND:Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of ...cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease.
METHODS:The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, Ldlr, based on a relevant familial hypercholesterolemia–related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into Ldlr mice.
RESULTS:We found that homogeneous Ldlr mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration.
CONCLUSIONS:Our work shows that in vivo AAV-CRISPR/Cas9–mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.
•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.
RATIONALE: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.
OBJECTIVE: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.
METHODS AND RESULTS: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 (CoEC-Cre) and a brain endothelial cell–specific Cre (BEC-Cre). Through lineage tracing, gene knockout and overexpression experiments, we demonstrated that CoEC-Cre and BEC-Cre efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of vascular endothelial growth factor receptor 2 using BEC-Cre, 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.
CONCLUSIONS: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.
BACKGROUND:Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The ...putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1, Bmi1, Isl1, or Abcg2 cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart.
METHODS:To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes.
RESULTS:By this stem cell marker–free and dual recombinases–mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury.
CONCLUSIONS:This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.
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.
RATIONALE:Endocardium is the major source of coronary endothelial cells (ECs) in the fetal and neonatal hearts. It remains unclear whether endocardium in the adult stage is also the main origin of ...neovascularization after cardiac injury.
OBJECTIVE:To define the vascular potential of adult endocardium in homeostasis and after cardiac injuries by fate-mapping studies.
METHODS AND RESULTS:We generate an inducible adult endocardial Cre line (Npr3 natriuretic peptide receptor C-CreER) and show that Npr3-CreER efficiently and specifically labels endocardial cells but not coronary blood vessels in the adult heart. The adult endocardial cells do not contribute to any vascular ECs during cardiac homeostasis. To examine the formation of blood vessels from endocardium after injury, we generate 4 cardiac injury models with Npr3-CreER micemyocardial infarction, myocardial ischemia–reperfusion, cryoinjury, and transverse aortic constriction. Lineage tracing experiments show that adult endocardium minimally contributes to coronary ECs after myocardial infarction. In the myocardial ischemia–reperfusion, cryoinjury, or transverse aortic constriction models, adult endocardial cells do not give rise to any vascular ECs, and they remain on the inner surface of myocardium that connects with lumen circulation. In the myocardial infarction model, very few endocardial cells are trapped in the infarct zone of myocardium shortly after ligation of coronary artery, indicating the involvement of endocardial entrapment during blood vessels formation. When these adult endocardial cells are relocated and trapped in the infarcted myocardium by transplantation or myocardial constriction model, very few endocardial cells survive and gain vascular EC properties, and their contribution to neovascularization in the injured myocardium remains minimal.
CONCLUSIONS:Unlike its fetal or neonatal counterpart, adult endocardium naturally generates minimal, if any, coronary arteries or vascular ECs during cardiac homeostasis or after injuries.
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).
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