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After severe heart injury, fibroblasts are activated and proliferate excessively to form scar, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous in respect of their degree of activation, proliferation, and function during cardiac fibrosis. Here, by dual recombinases-mediated genetic lineage tracing, we found that endocardium-derived fibroblasts (EndoFb) preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, the pattern of which mirrors that of EndoFb distribution in the heart. Specific ablation of EndoFb alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of EndoFb during cardiac remodeling. Our study identified EndoFb as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury, and as a potential therapeutic target against cardiac fibrosis.

Liver fibrogenesis is a highly dynamic and complex process that drives the progression of chronic liver disease toward liver failure and end-stage liver diseases. Despite decades of intense studies, the cellular and molecular mechanisms underlying liver fibrogenesis remain elusive, and no approved therapies to treat liver fibrosis are currently available. The rapid development of single-cell RNA sequencing (scRNA-seq) technologies allows the characterization of cellular alterations under healthy and diseased conditions at an unprecedented resolution. In this Review, we discuss how the scRNA-seq studies are transforming our understanding of the regulatory mechanisms of liver fibrosis. We specifically emphasize discoveries on disease-relevant cell subpopulations, molecular events, and cell interactions on cell types including hepatocytes, liver sinusoidal endothelial cells, myofibroblasts, and macrophages. These discoveries have uncovered critical pathophysiological changes during liver fibrogenesis. Further efforts are urged to fully understand the functional contributions of these changes to liver fibrogenesis, and to translate the new knowledge into effective therapeutic approaches.

Hematopoiesis is a highly orchestrated biological process sustaining the supply of leukocytes involved in the maintenance of immunity, O2 and CO2 exchange, and wound healing throughout the life-time of an animal including human. During early hematopoietic cell development, several waves of hematopoiesis require the precise regulation of hematopoietic ontogeny as well as the maintenance of hematopoietic stem and progenitor cells (HSPCs) in the hematopoietic tissues, such as the fetal liver and bone marrow (BM). Recently, emerging evidence has suggested the critical role of m6A mRNA modification, an epigenetic modification dynamically regulated by its effector proteins, in the generation and maintenance of hematopoietic cells during embryogenesis. In the adulthood, m6A has also been demonstrated to be involved in the functional maintenance of HSPCs in the BM and umbilical cord blood, as well as the progression of malignant hematopoiesis. In this review, we focus on recent progress in identifying the biological functions of m6A mRNA modification, its regulators and downstream gene targets during normal and pathological hematopoiesis. We propose that targeting m6A mRNA modification could offer novel insights into therapeutic development against abnormal and malignant hematopoietic cell development in the future.

Precisely deciphering the cellular plasticity in vivo is essential in understanding many key biological processes. Site-specific recombinases are genetic tools used for in vivo lineage tracing and gene manipulation. Conventional Cre-loxP, Dre-rox, and Flp-frt technologies form the orthogonal recombination systems that can also be used in combination to increase the precision. As such, more than one marker gene can be targeted for lineage tracing, studying cellular heterogeneity, recording cellular activities, or even genome editing. Their combinatory use has recently resolved some controversies in defining cellular fate plasticity. Focusing on cell fate studies, we introduce the design principles of orthogonal recombinases-based strategies, describe some working examples in resolving cell fate-related controversies, and discuss some of their technical strengths and limits.

The global propagation of SARS-CoV-2 leads to an unprecedented public health emergency. Despite that the lungs are the primary organ targeted by COVID-19, systemic endothelial inflammation and dysfunction is observed particularly in patients with severe COVID-19, manifested by elevated endothelial injury markers, endotheliitis, and coagulopathy. Here, we review the clinical characteristics of COVID-19 associated endothelial dysfunction; and the likely pathological mechanisms underlying the disease including direct cell entry or indirect immune overreactions after SARS-CoV-2 infection. In addition, we discuss potential biomarkers that might indicate the disease severity, particularly related to the abnormal development of thrombosis that is a fatal vascular complication of severe COVID-19. Furthermore, we summarize clinical trials targeting the direct and indirect pathological pathways after SARS-CoV-2 infection to prevent or inhibit the virus induced endothelial disorders., Graphical abstract Unlabelled Image

To date, identification of nonislet-specific transcriptional factors in the regulation of insulin gene expression has been little studied. Here, we report that the expression level of the transcription factor YY1 is increased dramatically in both human and mouse pancreatic β-cells after birth. Nevertheless, the physiological role of YY1 during β-cell development and its regulatory mechanism in β-cell function remain largely unknown. After β-cell ablation of Yy1, we observed rapid onset of hyperglycemia, impaired glucose tolerance, and reduced β-cell mass in neonatal and adult mice. These mice also had hypoinsulinemia with normal insulin sensitivity compared with their wild-type littermates, manifesting as a type 1 diabetic phenotype. Mechanistically, genome-wide RNA sequencing has defined dysregulated insulin signaling and defective glucose responsiveness in β-cells devoid of YY1. Integrative analyses coupled with chromatin immunoprecipitation assays targeting YY1, and histone modifications, including H3K4me1, H3K27ac, and H3K27me3, have further identified Ins1 and Ins2 as direct gene targets of YY1. Luciferase reporter assays and loss- and gain-of-function experiments also demonstrated that YY1 binds to the enhancer regions in exon 2 of Ins1 and Ins2, activating insulin transcription and, therefore, proinsulin and insulin production in pancreatic β-cells. YY1 also directly interacts with RNA polymerase II, potentially stabilizing the enhancer-promoter interaction in the multiprotein-DNA complex during transcription initiation. Taken together, our findings suggest a role for YY1 as a transcriptional activator of insulin gene expression, assisting β-cell maturation and function after birth. These analyses may advance our understanding of β-cell biology and provide clinically relevant insights targeting the pathophysiological origins of diabetes.

Background: Cardiac fibrosis is a lethal outcome of excessive formation of myofibroblasts that are scar-forming cells accumulated after heart injury. It has been reported that cardiac endothelial cells (ECs) contribute to a substantial portion of myofibroblasts through endothelial to mesenchymal transition (EndoMT). Recent lineage tracing studies demonstrate that myofibroblasts are derived from the expansion of resident fibroblasts rather than from the transdifferentiation of ECs. However, it remains unknown whether ECs can transdifferentiate into myofibroblasts reversibly or EndoMT genes were just transiently activated in ECs during cardiac fibrosis. Methods: By using the dual recombination technology based on Cre-loxP and Dre-rox, we generated a genetic lineage tracing system for tracking EndoMT in cardiac ECs. We used it to examine if there is transiently activated mesenchymal gene expression in ECs during cardiac fibrosis. Activation of the broadly used marker gene in myofibroblasts, αSMA (α-smooth muscle actin), and the transcription factor that induces epithelial to mesenchymal transition, Zeb1 (zinc finger E-box–binding homeobox 1), was examined. Results: The genetic system enables continuous tracing of transcriptional activity of targeted genes in vivo. Our genetic fate mapping results revealed that a subset of cardiac ECs transiently expressed αSMA and Zeb1 during embryonic valve formation and transdifferentiated into mesenchymal cells through EndoMT. Nonetheless, they did not contribute to myofibroblasts, nor transiently expressed αSMA or Zeb1 after heart injury. Instead, expression of αSMA was activated in resident fibroblasts during cardiac fibrosis. Conclusions: Mesenchymal gene expression is activated in cardiac ECs through EndoMT in the developing heart, but ECs do not transdifferentiate into myofibroblasts, nor transiently express some known mesenchymal genes during homeostasis and fibrosis in the adult heart. Resident fibroblasts that are converted to myofibroblasts by activating mesenchymal gene expression are the major contributors to cardiac fibrosis.

Unraveling cell–cell interaction is fundamental to understanding many biological processes. To date, genetic tools for labeling neighboring cells in mammals are not available. Here, we developed a labeling strategy based on the Cre-induced intercellular labeling protein (CILP). Cre-expressing donor cells release a lipid-soluble and membrane-permeable fluorescent protein that is then taken up by recipient cells, enabling fluorescent labeling of neighboring cells. Using CILP, we specifically labeled endothelial cells surrounding a special population of hepatocytes in adult mice and revealed their distinct gene signatures. Our results highlight the potential of CILP as a platform to reveal cell–cell interactions and communications in vivo.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

Fundació Carreras Liwei Lu dedicates this work to his mentor Dr Dennis G. Osmond for his pioneering work in B-cell biology. This work was supported by Chongqing International Institute for Immunology (2020YJC10), National Natural Science Foundation of China (82071817, 91842304, 82171771, and 82271854), Shenzhen Science and Technology Program (JCYJ20210324114602008), Hong Kong Research Grants Council (17113319 and 17103821), RGC Theme-based Research Scheme (TRS) (T12-703/19-R), and the Centre for Oncology and Immunology under the Health@InnoHK Initiative funded by the Innovation and Technology Commission, Hong Kong, China. The figures were created with BioRender.com. B cells play a pivotal role in the pathogenesis of autoimmune diseases. Although previous studies have shown many genetic polymorphisms associated with B-cell activation in patients with various autoimmune disorders, progress in epigenetic research has revealed new mechanisms leading to B-cell hyperactivation. Epigenetic mechanisms, including those involving histone modifications, DNA methylation, and noncoding RNAs, regulate B-cell responses, and their dysregulation can contribute to the pathogenesis of autoimmune diseases. Patients with autoimmune diseases show epigenetic alterations that lead to the initiation and perpetuation of autoimmune inflammation. Moreover, many clinical and animal model studies have shown the promising potential of epigenetic therapies for patients. In this review, we present an up-to-date overview of epigenetic mechanisms with a focus on their roles in regulating functional B-cell subsets. Furthermore, we discuss epigenetic dysregulation in B cells and highlight its contribution to the development of autoimmune diseases. Based on clinical and preclinical evidence, we discuss novel epigenetic biomarkers and therapies for patients with autoimmune disorders.

Summary Ductal cells have been proposed as a source of adult β cell neogenesis, but this has remained controversial. By combining lineage tracing, 3D imaging, and single-cell RNA sequencing (scRNA-seq) approaches, we show that ductal cells contribute to the β cell population over time. Lineage tracing using the Neurogenin3 (Ngn3)-CreERT line identified ductal cells expressing the endocrine master transcription factor Ngn3 that were positive for the δ cell marker somatostatin and occasionally co-expressed insulin. The number of hormone-expressing ductal cells was increased in Akita+/− diabetic mice, and ngn3 heterozygosity accelerated diabetes onset. scRNA-seq of Ngn3 lineage-traced islet cells indicated that duct-derived somatostatin-expressing cells, some of which retained expression of ductal markers, gave rise to β cells. This study identified Ngn3-expressing ductal cells as a source of adult β cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to β cell proliferation, maintains the adult islet β cell population., Graphical abstract, Highlights • Ductal Ngn3+ cells contribute to the beta cell population during homeostasis • Duct-resident endocrine progenitor cells express somatostatin • Somatostatin-positive ductal cells are increased in Akita+/− diabetic mice • scRNA-seq analysis suggests ductal somatostatin+ cells give rise to beta cells, A better understanding of adult beta cell neogenesis would open new approaches for diabetes treatment. Gribben et al. used lineage tracing and scRNA-seq to characterize a duct-resident somatostatin-positive endocrine progenitor cell population that is a source of beta cells in homeostasis and during diabetes.

To date, identification of non-islet specific transcriptional factors in the regulation of insulin gene expression has been less studied. Here, we report that the expression level of the transcription factor YY1 has increased dramatically after birth in both human and mouse pancreatic beta cells. Nevertheless, the physiological role of YY1 during beta cell development, and its regulatory mechanism in beta cell function remain largely unknown. Following beta cell ablation of Yy1, we observed rapid onset of hyperglycemia, impaired glucose tolerance and reduced beta cell mass in both neonates and adult mice. These mice also displayed hypoinsulinemia with normal insulin sensitivity compared to their wildtype littermates, manifesting as a type-1 diabetic phenotype. Mechanistically, genome-wide RNA-seq has defined dysregulated insulin signalling and defective glucose responsiveness in beta cells devoid of YY1. Integrative analyses coupled with chromatin immunoprecipitation (ChIP) assays targeting YY1 and histone modifications including H3K4me1, H3K27ac and H3K27me3 have further identified Ins1 and Ins2 as direct gene targets of YY1. Further analyses through luciferase reporter assays, loss- and gain-of-function experiments demonstrate that YY1 binds to the enhancer regions in exon 2 of Ins1 and Ins2, activating insulin transcription and, therefore, proinsulin and insulin production in pancreatic beta cells. YY1 also directly interacts with RNA polymerase II, potentially stabilising the enhancer-promoter interaction in the multiprotein-DNA complex during transcription initiation. Taken together, our findings have suggested a hitherto novel role of YY1 as a transcriptional activator of insulin gene expression, assisting beta cell maturation and function after birth. These studies may advance our understanding of beta cell biology with clinically relevant insights targeting the pathophysiological origins of diabetes.

Vascular endothelium maintains vascular homeostasis through liberating a spectrum of vasoactive molecules, both protective and harmful regulators of vascular tone, structural remodeling, inflammation and atherogenesis. An intricate balance between endothelium-derived relaxing factors (nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor) and endothelium-derived contracting factors (superoxide anion, endothelin-1 and constrictive prostaglandins) tightly regulates vascular function. Disruption of such balance signifies endothelial dysfunction, a critical contributor in aging and chronic cardiometabolic disorders, such as obesity, diabetes, hypertension, dyslipidemia and atherosclerotic vascular diseases. Among many proposed cellular and molecular mechanisms causing endothelial dysfunction, oxidative stress and inflammation are often the pivotal players and they are naturally considered as useful targets for intervention in patients with cardiovascular and metabolic diseases. In this article, we provide a recent update on the therapeutic values of pharmacological agents, such as cyclooxygenase-2 inhibitors, renin-angiotensin-system inhibitors, bone morphogenic protein 4 inhibitors, peroxisome proliferator-activated receptor δ agonists, and glucagon-like peptide 1-elevating drugs, and the physiological factors, particularly hemodynamic forces, that improve endothelial function by targeting endothelial oxidative stress and inflammation.

Enhancement of protein synthesis from mRNA translation is one of the key steps supporting cardiomyocyte hypertrophy during cardiac remodeling. The methyltransferase-like5 (METTL5), which catalyzes m

With the continuous development of RNA biology and massive genome-wide transcriptome analysis, more and more RNA molecules and their functions have been explored in the last decade. Increasing evidence has demonstrated that RNA-related regulatory networks play an important role in a variety of human diseases, including cardiovascular diseases. In this review, we focus on RNA regulatory networks in heart disease, most of which are devastating conditions with no known cure. We systemically summarize recent discoveries of important new components of RNA regulatory networks, including microRNAs, long non-coding RNAs, and circular RNAs, as well as multiple regulators that affect the activity of these networks in cardiac physiology and pathology. In addition, this review covers emerging micropeptides, which represent short open reading frames (sORFs) in long non-coding RNA transcripts that may modulate cardiac physiology. Based on the current knowledge of RNA regulatory networks, we think that ongoing discoveries will not only provide us a better understanding of the molecular mechanisms that underlie heart disease, but will also identify novel biomarkers and therapeutic targets for the diagnosis and treatment of cardiac disease., Graphical Abstract, Heart disease is the leading cause of death around the world with no known cure in many cases. Recent discoveries of important new components of RNA regulatory networks in cardiac pathology reviewed herein identify potential novel biomarkers and therapeutic targets for the diagnosis and treatment of this devastating disease.

Unlike adult cardiomyocytes, neonatal cardiomyocytes can readily proliferate that contributes to a transient regenerative potential after myocardial injury in mice. We have recently reported that CD4+ regulatory T-cells promote this process; however, the role of other CD4+ T-cell subsets as well as CD8+ T-cells in postnatal heart regeneration has been less studied. Methods: by comparing the regenerating postnatal day (P) 3 and the non-regenerating P8 heart after injury, we revealed the heterogeneity of CD4+ and CD8+ T-cells in the myocardium through single cell analysis. We also specifically ablated CD4+ and CD8+ T-cells using the lytic anti-CD4 and -CD8 monoclonal antibodies, respectively, in juvenile mice at P8 after myocardial injury. Results: we observe significantly more CD4+FOXP3- conventional T-cells in the P8 heart when compared to that of the P3 heart within a week after injury. Surprisingly, such a difference is not seen in CD8+ T-cells that appear to have no function as their depletion does not reactivate heart regeneration. On the other hand, specific ablation of CD4+ T-cells contributes to mitigated cardiac fibrosis and increased cardiomyocyte proliferation after injury in juvenile mice. Single-cell transcriptomic profiling reveals a pro-fibrotic CD4+ T-cell subset in the P8 but not P3 heart. Moreover, there are likely more Th1 and Th17 cells in the P8 than P3 heart. We further demonstrate that cytokines of Th1 and Th17 cells can directly reduce the proliferation and increase the apoptosis of neonatal cardiomyocytes. Moreover, ablation of CD4+ T-cells can directly or indirectly facilitate the polarization of macrophages away from the pro-fibrotic M2-like signature in the juvenile heart. Nevertheless, ablation of CD4+ T-cells alone does not offer the same protection in the adult heart after myocardial infarction, suggesting a developmental change of immune cells including CD4+ T-cells in the regulation of age-related mammalian heart repair. Conclusions: our results demonstrate that ablation of CD4+ but not CD8+ T-cells promotes heart regeneration in juvenile mice; and CD4+ T-cells play a distinct role in the regulation of heart regeneration and repair during development.

Accumulating evidence has demonstrated that immune cells play an important role in the regulation of tissue repair and regeneration. After injury, danger signals released by the damaged tissue trigger the initial pro-inflammatory phase essential for removing pathogens or cellular debris that is later replaced by the anti-inflammatory phase responsible for tissue healing. On the other hand, impaired immune regulation can lead to excessive scarring and fibrosis that could be detrimental for the restoration of organ function. Regulatory T-cells (Treg) have been revealed as the master regulator of the immune system that have both the immune and regenerative functions. In this review, we will summarize their immune role in the induction and maintenance of self-tolerance; as well as their regenerative role in directing tissue specific response for repair and regeneration. The latter is clearly demonstrated when Treg enhance the differentiation of stem or progenitor cells such as satellite cells to replace the damaged skeletal muscle, as well as the proliferation of parenchymal cells including neonatal cardiomyocytes for functional regeneration. Moreover, we will also discuss the reparative and regenerative role of Treg with a particular focus on blood vessels and cardiac tissues. Last but not least, we will describe the ongoing clinical trials with Treg in the treatment of autoimmune diseases that could give clinically relevant insights into the development of Treg therapy targeting tissue repair and regeneration.

In this study, we observe that the ischemic tissues of type-2 diabetic (T2D) patients and mice have significantly more CD8+ T-cells than that of their normoglycemic counterparts, respectively. However, the role of CD8+ T-cells in the pathogenesis of diabetic vascular complication has been less studied. Methods: We employed loss-of-function studies in mouse models using the non-lytic anti-CD8 antibody that blocks tissue infiltration of CD8+ T-cells into the injured tissue. We also performed genome-wide, single-cell RNA-sequencing of CD8+ T-cells to uncover their role in the pathogenesis of diabetic vascular diseases. Results: The vascular density is negatively correlated with the number of CD8+ T-cells in the ischemic tissues of patients and mice after injury. CD8+ T-cells or their supernatant can directly impair human and murine angiogenesis. Compared to normoglycemic mice that can regenerate their blood vessels after injury, T2D mice fail in this regeneration. Treatment with the CD8 checkpoint blocking antibody increases the proliferation and function of endothelial cells in both Leprdb/db mice and diet-induced diabetic Cdh5-Cre;Rosa-YFP lineage-tracing mice after ischemic injury. Furthermore, single-cell transcriptomic profiling reveals that CD8+ T-cells of T2D mice showed a de novo cell fate change from the angiogenic, tissue-resident memory cells towards the effector and effector memory cells after injury. Functional revascularization by CD8 checkpoint blockade is mediated through unleashing such a poised lineage commitment of CD8+ T-cells from T2D mice. Conclusion: Our results reveal that CD8+ T-cell plasticity regulates vascular regeneration; and give clinically relevant insights into the potential development of immunotherapy targeting vascular diseases associated with obesity and diabetes.

RATIONALE: Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) protects against myocardial ischemia-reperfusion injury (I/R). However, reperfusion of myocardium results in superoxide (O(2)(•−)) generation, which promotes eNOS glutathionylation that produces O(2)(•−) instead of NO. It is unclear whether glutathionylated eNOS (SG-eNOS) continues to produce O(2)(•−) indefinitely or undergoes a time-dependent degradation. OBJECTIVE: To determine whether SG-eNOS continues to produce O(2)(•−) in I/R for a prolonged period causing accentuated I/R injury or it undergoes a time-dependent degradation. METHODS AND RESULTS: Since SG-eNOS produces significant O(2)(•−) instead of NO, we sought to determine the time-course of SG-eNOS levels in the HCAEC in hypoxia/reoxygenation (H/R) by western analysis and immunoprecipitation. SG-eNOS was degraded by chaperone-mediated autophagy (CMA), as inhibitors of CMA rescued eNOS expression. We further confirmed CMA by high resolution confocal and electron microscopy. We showed that SG-eNOS is targeted by HSC70 chaperone via its interaction with glutathionylated-cysteine 691 and 910. Glutathionylation of cysteine 691 residue in H/R exposes 735QRYRL739 motif for interaction with HSC70, and consequent transportation to LAMP2A vesicle, where it is degraded by lysosomal proteases. Mutagenesis of these residues in eNOS inhibited its CMA. Using contrast echocardiography and electron paramagnetic resonance spectrometry (EPR), we found that Trx-Tg mice show improved myocardial perfusion and decreased myocardial apoptosis in I/R due to deglutathionylation of SG-eNOS and restoration of NO generation. Further, WT mice treated with recombinant human Trx (rhTrx) were protected against eNOS CMA, and restored NO production with improved myocardial perfusion and decreased I/R injury. CONCLUSIONS: SG-eNOS undergoes degradation via CMA, following prolonged retention in the cytosol. CMA of SG-eNOS terminates O(2)(•−) generation preventing further tissue damage but causes irreversible loss of eNOS and NO availability. Prompt deglutathionylation of SG-eNOS prevents CMA, promotes NO production, and improved myocardial perfusion, resulting in amelioration of reperfusion injury.

Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.

Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1β (IL-1β) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1β axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.

Rationale: The developing heart is composed of cardiomyocytes and noncardiomyocytes since the early stage. It is generally believed that noncardiomyocytes including the cardiac progenitors contribute to new cardiomyocytes of the looping heart. However, it remains unclear what the cellular dynamics of nonmyocyte to cardiomyocyte conversion are and when the lineage segregation occurs during development. It also remains unknown whether nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. Objective: We quantify the lineage conversion of noncardiomyocytes to cardiomyocytes in the embryonic and neonatal hearts and determine when the 2 cell lineages segregate during heart development. Moreover, we directly test if nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. Methods and Results: We generated a dual genetic lineage tracing strategy in which cardiomyocytes and noncardiomyocytes of the developing heart could be simultaneously labeled by 2 orthogonal recombination systems. Genetic fate mapping showed that nonmyocyte to cardiomyocyte conversion peaks at E8.0 (embryonic day) to E8.5 and gradually declines at E9.5 and E10.5. Noncardiomyocytes do not generate any cardiomyocyte at and beyond E11.5 to E12.5. In the neonatal heart, noncardiomyocytes also do not contribute to any new cardiomyocyte in homeostasis or after injury. Conclusions: Noncardiomyocytes contribute to new cardiomyocytes of the developing heart at early embryonic stage before E11.5. The noncardiomyocyte and cardiomyocyte lineage segregation occurs between E10.5 and E11.5, which is maintained afterward even during neonatal heart regeneration.

Summary Elucidation of the role of different cell lineages in the liver could offer avenues to drive liver regeneration. Previous studies showed that SOX9+ hepatocytes can differentiate into ductal cells after liver injuries. It is unclear whether SOX9+ hepatocytes are uni- or bipotent progenitors at a single-cell level during liver injury. Here, we developed a genetic tracing system to delineate the lineage potential of SOX9+ hepatocytes during liver homeostasis and regeneration. Fate-mapping data showed that these SOX9+ hepatocytes respond specifically to different liver injuries, with some contributing to a substantial number of ductal cells. Clonal analysis demonstrated that a single SOX9+ hepatocyte gives rise to both hepatocytes and ductal cells after liver injury. This study provides direct evidence that SOX9+ hepatocytes can serve as bipotent progenitors after liver injury, producing both hepatocytes and ductal cells for liver repair and regeneration., Graphical Abstract, Highlights • SOX9+ hepatocytes respond distinctly to different liver injuries • Generation of a Confetti reporter responsive to dual recombinases • SOX9+ hepatocytes can serve as bipotent progenitors after liver injury, In this article, Zhou and colleagues developed an intersectional genetic strategy to specifically label SOX9+ hepatocytes. Using a dual recombinase-mediated Confetti reporter, they showed that a subset of SOX9+ hepatocytes, at single-cell level, are bipotent progenitors that differentiate into both hepatocytes and biliary epithelial cells during liver injury and repair.

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato

It has been suggested that new beta cells can arise from specific populations of adult pancreatic progenitors or facultative stem cells. However, their existence remains controversial, and the conditions under which they would contribute to new beta-cell formation are not clear. Here, we use a suite of mouse models enabling dual-recombinase-mediated genetic tracing to simultaneously fate map insulin-positive and insulin-negative cells in the adult pancreas. We find that the insulin-negative cells, of both endocrine and exocrine origin, do not generate new beta cells in the adult pancreas during homeostasis, pregnancy or injury, including partial pancreatectomy, pancreatic duct ligation or beta-cell ablation with streptozotocin. However, non-beta cells can give rise to insulin-positive cells after extreme genetic ablation of beta cells, consistent with transdifferentiation. Together, our data indicate that pancreatic endocrine and exocrine progenitor cells do not contribute to new beta-cell formation in the adult mouse pancreas under physiological conditions. Zhao et al. use genetic lineage tracing to demonstrate that pancreatic endocrine and exocrine progenitor cells do not generate new beta cells, thus arguing against beta-cell neogenesis in the adult mouse pancreas.

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

Distraction osteogenesis (DO) is a well-established surgical technique for treating bone defect and limb lengthening. The major drawback of DO is the long treatment period as the external fixator has to be kept in place until consolidation is completed. Calcitonin gene-related peptide (CGRP) has been reported to promote angiogenesis by affecting endothelial progenitor cells (EPCs) in limb ischemia and wound healing. Thus, the goal of this study was to evaluate the angiogenic effect of exogenous CGRP on bone regeneration in a rat DO model. Exogenous CGRP was directly injected into the bone defect after each cycle of distraction in vivo. Microcomputed tomography, biomechanical test, and histological analysis were performed to assess the new bone formation. Angiography and immunofluorescence were performed to assess the formation of blood vessels. CD31+CD144+ EPCs in the bone defect were quantified with flow cytometry. In in vitro study, bone marrow stem cells (BMSCs) were used to investigate the effect of CGRP on EPCs production during endothelial differentiation. Our results showed that CGRP significantly promoted bone regeneration and vessel formation after consolidation. CGRP significantly increased the fraction of CD31+CD144+EPCs and the capillary density in the bone defect at the end of distraction phase. CGRP increased EPC population in the endothelial differentiation of BMSCs in vitro by activating PI3K/AKT signaling pathway. Furthermore, differentiated EPCs rapidly assembled into tube-like structures and promoted osteogenic differentiation of BMSCs. In conclusion, CGRP increased EPC population and promoted blood vessel formation and bone regeneration at the defect region in a DO model. Impact statement Distraction osteogenesis (DO) is a well-established surgical technique for limb lengthening and bone defect. The disadvantage of this technique is that external fixator is needed to be kept in place for about 12 months. This may result in increased risk of infection, financial burden, and negative psychological impacts. In this study, we have injected calcitonin gene-related peptide (CGRP) into the defect region after distraction and found that CGRP enhanced vessel formation and bone regeneration in a rat DO model. This suggests that a controlled delivery system for CGRP could be developed and applied clinically for accelerating bone regeneration in patients with DO.

Unraveling the fates of resident stem cells during tissue regeneration is an important objective in clinical and basic research. Genetic lineage tracing based on Cre–loxP recombination provides an effective strategy for inferring cell fate and cell conversion in vivo. However, the determination of the exact fates of resident stem cells or their derivatives in disease states and during tissue regeneration remains controversial in many fields of study, partly because of technical limitations associated with Cre-based lineage tracing, such as, for example, off-target labeling. Recently, we generated a new lineage-tracing platform we named DeaLT (dual-recombinase-activated lineage tracing) that uses the Dre–rox recombination system to enhance the precision of Cre-mediated lineage tracing. Here, we describe as an example a detailed protocol using DeaLT to trace the fate of c-Kit+ cardiac stem cells and their derivatives, in the absence of any interference from nontarget cells such as cardiomyocytes, during organ homeostasis and after tissue injury. This lineage-tracing protocol can also be used to delineate the fate of resident stem cells of other organ systems, and takes ~10 months to complete, from mouse crossing to final tissue analysis. In this protocol, the authors explain a new, more precise genetic-lineage-tracing system based on a dual-recombinase strategy. DeaLT enables specific fate mapping of resident stem cells by using both the Cre–loxP and Dre–rox systems.