Kathy O. Lui

Endocardium Contributes to Cardiac Fat

RATIONALEnUnraveling the developmental origin of cardiac fat could offer important implications for the treatment of cardiovascular disease. The recent identification of the mesothelial source of epicardial fat tissues reveals a heterogeneous origin of adipocytes in the adult heart. However, the developmental origin of adipocytes inside the heart, namely intramyocardial adipocytes, remains largely unknown.nnnOBJECTIVEnTo trace the developmental origin of intramyocardial adipocytes.nnnMETHODS AND RESULTSnIn this study, we identified that the majority of intramyocardial adipocytes were restricted to myocardial regions in close proximity to the endocardium. Using a genetic lineage tracing model of endocardial cells, we found that Nfatc1(+) endocardial cells contributed to a substantial number of intramyocardial adipocytes. Despite the capability of the endocardium to generate coronary vascular endothelial cells surrounding the intramyocardial adipocytes, results from our lineage tracing analyses showed that intramyocardial adipocytes were not derived from coronary vessels. Nevertheless, the endocardium of the postnatal heart did not contribute to intramyocardial adipocytes during homeostasis or after myocardial infarction.nnnCONCLUSIONSnOur in vivo fate-mapping studies demonstrated that the developing endocardium, but not the vascular endothelial cells, gives rise to intramyocardial adipocytes in the adult heart.

GATA4 regulates Fgf16 to promote heart repair after injury

Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair. Highlighted article: GATA4 and FGF16 are important mediators of cardiomyocyte proliferation and hypertrophy during neonatal heart repair following cryoinjury and apex resection.

Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics

Significance The human placenta is a dynamic and cellular heterogeneous organ, which is critical in fetomaternal homeostasis and the development of preeclampsia. Previous work has shown that placenta-derived cell-free RNA increases during pregnancy. We applied large-scale microfluidic single-cell transcriptomic technology to comprehensively characterize cellular heterogeneity of the human placentas and identified multiple placental cell-type–specific gene signatures. Analysis of the cellular signature expression in maternal plasma enabled noninvasive delineation of the cellular dynamics of the placenta during pregnancy and the elucidation of extravillous trophoblastic dysfunction in early preeclampsia. The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.

Regulatory T-Cells: Potential Regulator of Tissue Repair and Regeneration

The identification of stem cells and growth factors as well as the development of biomaterials hold great promise for regenerative medicine applications. However, the therapeutic efficacy of regenerative therapies can be greatly influenced by the host immune system, which plays a pivotal role during tissue repair and regeneration. Therefore, understanding how the immune system modulates tissue healing is critical to design efficient regenerative strategies. While the innate immune system is well known to be involved in the tissue healing process, the adaptive immune system has recently emerged as a key player. T-cells, in particular, regulatory T-cells (Treg), have been shown to promote repair and regeneration of various organ systems. In this review, we discuss the mechanisms by which Treg participate in the repair and regeneration of skeletal and heart muscle, skin, lung, bone, and the central nervous system.

Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases

Endothelial cells and mesenchymal cells are two different cell types with distinct morphologies, phenotypes, functions, and gene profiles. Accumulating evidence, notably from lineage-tracing studies, indicates that the two cell types convert into each other during cardiovascular development and pathogenesis. During heart development, endothelial cells transdifferentiate into mesenchymal cells in the endocardial cushion through endothelial-to-mesenchymal transition (EndoMT), a process that is critical for the formation of cardiac valves. Studies have also reported that EndoMT contributes to the development of various cardiovascular diseases, including myocardial infarction, cardiac fibrosis, valve calcification, endocardial elastofibrosis, atherosclerosis, and pulmonary arterial hypertension. Conversely, cardiac fibroblasts can transdifferentiate into endothelial cells and contribute to neovascularization after cardiac injury. However, progress in genetic lineage tracing has challenged the role of EndoMT, or its reversed programme, in the development of cardiovascular diseases. In this Review, we discuss the caveats of using genetic lineage-tracing technology to investigate cell-lineage conversion; we also reassess the role of EndoMT in cardiovascular development and diseases and elaborate on the molecular signals that orchestrate EndoMT in pathophysiological processes. Understanding the role and mechanisms of EndoMT in diseases will unravel the therapeutic potential of targeting this process and will provide a new paradigm for the development of regenerative medicine to treat cardiovascular diseases.Endothelial-to-mesenchymal transition (EndoMT) is critical for the formation of the cardiac valves and contributes postnatally to the development of cardiovascular diseases. However, progress in lineage-tracing technology has challenged the role of EndoMT in cardiac fibrosis. In this Review, Li and colleagues discuss the caveats of using lineage tracing to investigate cell-lineage conversion and reassess the role of EndoMT in cardiovascular development and diseases.Key pointsEndothelial cells are converted to mesenchymal cells through endothelial-to-mesenchymal transition (EndoMT) during cardiovascular development.EndoMT also has a critical role in the development of cardiovascular diseases, including cardiac valve diseases, tissue fibrosis, pulmonary arterial hypertension, and atherosclerosis.The involvement of EndoMT in fibroblast contribution during cardiac fibrosis is an ongoing debate.Lineage-tracing studies indicate that resident fibroblasts, rather than EndoMT, give rise to the majority of myofibroblasts after injury.Pre-existing endothelial cells, but not mesenchymal-to-endothelial transition, are the main source that mediates neovascularization after cardiac injury.

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases

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.

Regulatory T Cells Promote Apelin-Mediated Sprouting Angiogenesis in Type 2 Diabetes

The role of CD4+ Txa0cells in the ischemic tissues of T2D patients remains unclear. Here, we report that T2D patients vascular density was negatively correlated with the number of infiltrating CD4+ Txa0cells after ischemic injury. Th1 was the predominant subset, and Th1-derived IFN-γ and TNF-α directly impaired human angiogenesis. We then blocked CD4+ Txa0cell infiltration into the ischemic tissues of both Leprdb/db and diet-induced obese T2D mice. Genome-wide RNA sequencing shows an increased proliferative and angiogenic capability of diabetic ECs in ischemic tissues. Moreover, wire myography shows enhanced EC function and laser Doppler imaging reveals improved post-ischemic blood reperfusion. Mechanistically, functional revascularization after CD4 coreceptor blockade was mediated by Tregs. Genetic lineage tracing via Cdh5-CreER and Apln-CreER and coculture assays further illustrate that Tregs increased vascular density and induced de novo sprouting angiogenesis in a paracrine manner. Taken together, our results reveal that Th1 impaired while Tregs promoted functional post-ischemic revascularization in obesity and diabetes.

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

Vascular Development and Regeneration in the Mammalian Heart

Cardiovascular diseases including coronary artery disease are the leading cause of death worldwide. Unraveling the developmental origin of coronary vessels could offer important therapeutic implications for treatment of cardiovascular diseases. The recent identification of the endocardial source of coronary vessels reveals a heterogeneous origin of coronary arteries in the adult heart. In this review, we will highlight recent advances in finding the sources of coronary vessels in the mammalian heart from lineage-tracing models as well as differentiation studies using pluripotent stem cells. Moreover, we will also discuss how we induce neovascularization in the damaged heart through transient yet highly efficient expression of VEGF-modified mRNAs as a potentially therapeutic delivery platform.

Regulatory T-cells are required for neonatal heart regeneration

Previous work has elegantly demonstrated that, unlike adult mammalian heart, the neonatal heart is able to regenerate after injury from postnatal day (P) 1 to 7. Recently, macrophages are found to be required in the repair process as depletion of which abolishes endogenous regenerative capability of the neonatal heart. Nevertheless, whether innate immunity alone is sufficient for neonatal heart regeneration is obscure. Here, we investigate a hitherto novel role of FOXP3+ regulatory T-cells (Treg) in neonatal heart regeneration. Unlike their wild type counterparts, NOD/SCID mice that are deficient for T-cells but innate immune cells including macrophages fail to regenerate their injured heart as early as P3. In wild type mice, both conventional CD4+ T-cells and Treg are recruited to cardiac muscle within the first week after injury. Treatment with the lytic anti-CD4 antibody that specifically depletes conventional CD4+ T-cells leads to reduced cardiac fibrosis; while treatment with the lytic anti-CD25 antibody that specifically depletes CD4+CD25hiFOXP3+ Treg contributes to increased fibrosis of the neonatal heart after injury. Moreover, adoptive transfer of Treg to NOD/SCID mice results in mitigated fibrosis and increased proliferation and function of cardiac muscle of the neonatal heart after injury. Mechanistically, single cell transcriptomic profiling reveals that Treg are a source of chemokines and cytokines that attract monocytes and macrophages previously known to drive neonatal heart regeneration. Furthermore, Treg directly promote proliferation of both mouse and human cardiomyocytes in a paracrine manner. Our findings uncover an unappreciated mechanism in neonatal heart regeneration; and offer new avenues for developing novel therapeutics targeting Treg-mediated heart regeneration.