Cell Proliferation

BACKGROUNDNuclear prelamin A recognition factor-like (NARFL) plays a crucial role in cytosolic iron-sulfur protein assembly (CIA) and protects cells against oxidative stress. In our previous study, we identified a novel homozygous mutation in NARFL that led to decreased expression in a consanguineous family with diffuse pulmonary arteriovenous malformations (DPAVMs) secondary to pulmonary hypertension. Additionally, we observed that narfl deletion in zebrafish resulted in larvae lethality, subintestinal vessel malformation, and increased oxidative stress. In this study, we aimed to further investigate the function of NARFL and elucidate the pathological manifestations of NARFL deficiency in zebrafish models, cellular models, mouse models, and clinical samples, focusing on the underlying molecular mechanisms. METHODSWe observed the behavioral and phenotypic abnormalities in zebrafish caused by narfl deletion and investigated the mechanism behind vascular morphological abnormalities. Furthermore, we constructed NARFL gene knockout stable cell lines in human pulmonary microvascular endothelial cells (HPMEC) to examine the morphological and functional changes in endothelial cells caused by NARFL deletion. We studied the effects of NARFL deletion on ferroptosis and its potential rescue using a ferroptosis inhibitor. To investigate the function of the human NARFL homolog Ciao3 gene in vascular development, we created a mouse model with a knockout of the Ciao3 gene. Finally, we compared the distribution of tagSNPs of NARFL using the SNaPshot method between cases and controls to confirm the role of the Ciao3 gene in endothelial dysfunction. RESULTSNarfl deletion in zebrafish resulted in larvae lethality, vascular malformation with abnormal blood flow, abnormal blood-brain barrier (BBB) structure, and brain neuron lesions. Fluorescence probe detection showed increased iron, enhanced oxidative stress, lipid peroxidation, and decreased mitochondrial respiration in response to narfl deficiency, which could be partially alleviated by the use of the ferroptosis inhibitor Ferrostatin-1. We observed downregulation of the iron-sulfur protein cyp2p8 expression in blood vessels of narfl-deficient zebrafish through qRT-PCR and WISH experiments. In HPMEC cells, NARFL deficiency resulted in decreased proliferation, abnormal mitochondrial morphology, increased levels of iron and oxidative stress, and decreased mitochondrial respiration. Functional experiments on endothelial cells revealed decreased tube formation ability and enhanced permeability in response to NARFL deficiency. WB experiments showed downregulation of GPX4, SLC7A11, and Ferritin, while TFR1 and IRP1 were upregulated. Downregulation of NARFL also affected the expression of the iron-sulfur protein CYP2J2. Co-IP results indicated that NARFL deletion led to incompatibility among the CIA system-associated proteins. In mice, Ciao3 deletion in the embryonic stage resulted in embryonic death, vascular dysplasia, impaired differentiation of endothelial progenitor cells, and abnormalities in the expression of ferroptosis-related proteins. Reduction of Ciao3 impaired vascular function and decreased ring formation ability in adult heterozygous mice. NARFL polymorphisms rs11248948, rs2071952, and rs611289 were identified as susceptible sites for epilepsy, while rs11792680 was associated with susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases. CONCLUSIONNARFL knockout disrupts its interaction with CIA system-related proteins, leading to decreased aconitase activity, increased IRP1 activity, endothelial cell ferroptosis pathway abnormalities, enhanced ferroptosis and oxidative stress, and ultimately vascular endothelial dysfunction. This dysfunction is responsible for the death of embryos in narfl-/- zebrafish and Ciao3-/- mice, as well as the susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases. What Is New?O_LIElucidation of the mechanism behind NARFL knockout-induced death through dynamic visualization experiments in vivo and mechanism and function experiments in vitro: The study explored the function of NARFL, as it is known as a "knockout lethal" protein. Both in vivo and in vitro experiments have confirmed that NARFL acts as the "transmitter" of cytoplasmic iron-sulfur clusters. Its absence prevents interaction with associated proteins of the CIA system, leading to reduced cisaconitase activity, enhanced IRP1 activity, ferroptosis of endothelial cells, and increased oxidative stress, eventually resulting in cell death. C_LIO_LIProviding new research ideas for the study of cytoplasmic iron-sulfur proteins: Most current studies focus on the function of mitochondrial iron-sulfur proteins and their relationship with iron death. However, research on extramitochondrial iron-sulfur proteins is relatively limited. This study provides data support and research ideas for understanding the function of extramitochondrial iron-sulfur proteins by exploring the pathological mechanism of NARFL and the mediation of iron-sulfur protein maturation. C_LI What Are the Clinical Implications?From rare diseases to common diseases: Through the investigation of the lethal mechanism of NARFL knockout and the study of NARFL gene polymorphisms associated with vascular endothelial dysfunction diseases, we propose the hypothesis that NARFL may be a susceptibility gene for these diseases. This study provides data support for the hypothesis and contributes to our understanding of the role of NARFL in vascular endothelial dysfunction diseases.

RationaleN6-Methyladenosine (m6A) is the most common type of RNA methylation modification, mainly occurring on mRNA. Whether m6A-modified circRNAs are involved in different settings of pulmonary fibrosis remains unclear. Methods and ResultsUsing an m6A-circRNA epitranscriptomic chip, candidate circRNAs were selected, in which hsa_circ_0000672 and hsa_circ_0005654 were specifically involved in SiO2-induced pulmonary fibrosis by targeting the same protein, eIF4A3, indicating that the m6A modification of these two circRNAs has a synergistic effect on fibroblast dysfunction induced by SiO2. A mechanistic study revealed that the m6A modification of circRNAs was mainly mediated by the methyltransferase METTL3. Furthermore, METTL3 promoted the activation, migration and activity of pulmonary fibroblasts and participated in SiO2-induced pulmonary fibrosis via circRNA m6A modification. Conclusionm6A methylation of circRNAs mediates silica-induced fibrosis via synergistic effects, enriching the understanding of circRNAs and uncovering a potential new target to treat fibrosis-related diseases.

The digestive system exhibits extensive diversity in developmental mechanisms and morphology across metazoans, yet the evolutionary origins underlying its organ differentiation remain unclear. Here, single-cell RNA sequencing was employed to investigate endodermal cell lineage specification during metamorphosis in the urochordate Styela clava, a newly established model for chordate evolution. By profiling 26,099 cells across five stages, we identified 21 major cell clusters and reconstructed the endodermal differentiation trajectories. Our analysis reveals two larval endodermal progenitor populations with distinct differentiation potentials. Pseudotime and RNA velocity analyses indicate that these progenitors give rise to stomach and intestinal lineages, respectively. Cross-species comparisons reveal putative homologous relationships between ascidian endodermal lineages and mouse definitive and visceral endodermal lineages, suggesting dual origins of digestive tract in chordates. We also identified conserved TGF-{beta} and FGF regulatory programs in digestive organ patterning and highlight earlier fate restriction of stomach and intestinal progenitors in ascidians compared to vertebrates. These findings provide insights into how chordate digestive organs evolved from ancestral endodermal patterning programs.

Maternal drug exposure during pregnancy increases the risks of developmental cardiotoxicity, leading to congenital heart defects (CHDs). In this study, we used human stem cells as an in-vitro system to interrogate the mechanisms underlying drug-induced toxicity during cardiomyocyte differentiation, including anticancer tyrosine kinase inhibitor (TKI) drugs (imatinib, sunitinib, and vandetanib). H1-ESCs were treated with these drugs at sublethal levels during cardiomyocyte differentiation. We found that early exposure to TKIs during differentiation induced obvious toxic effects in differentiated cardiomyocytes, including disarranged sarcomere structure, interrupted Ca2+-handling, and impaired mitochondrial function. As sunitinib exposure showed the most significant developmental cardiotoxicity of all TKIs, we further examine its effect with in-vivo experiments. Maternal sunitinib exposure caused fetal death, bioaccumulation, and histopathologic changes in the neonatal mice. Integrative analysis of both transcriptomic and chromatin accessibility landscapes revealed that TKI-exposure altered GATA4-mediated regulatory network, which included key mitochondrial genes. Overexpression of GATA4 with CRISPR-activation restored morphologies, contraction, and mitochondria function in cardiomyocytes upon TKI exposure early during differentiation. Altogether, our study identified a novel crosstalk mechanism between GATA4 activity and mitochondrial function during cardiomyocyte differentiation, and revealed potential therapeutic approaches for reducing TKI-induced developmental cardiotoxicity for human health. HighlightsO_LIEarly-stage exposure to TKIs induced cardiotoxicity and mitochondrial dysfunction C_LIO_LIGATA4 transcriptional activity is inhibited by TKIs C_LIO_LINetwork analysis reveals interactions between GATA4 and mitochondrial genes C_LIO_LIGATA4-overexpression rescues cardiomyocytes and mitochondria from TKI exposure C_LI

PFOA (perfluorooctanoic acid) is a difficult-to-degrade chemical that poses significant risks to human health and the environment. Studies have shown that PFOA affect female reproduction, but effect and mechanism of low doses PFOA expose on endometrial receptivity are unclear. In this study, we found that exposure to low doses of PFOA damaged endometrial receptivity in mice, resulting in decreased embryo implantation rates in mice. Furthermore, using single-cell sequencing technology, we systematically analyzed the specific mechanisms by which PFOA damages endometrial epithelial cell function and the ANGTL signaling pathway between endometrial stromal cells and epithelial cells, leading to embryo implantation failure. The elucidation of this mechanism provides new targets for the treatment of infertility about exposed to PFOA.

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC pluripotency since knockdown of Npac causes mouse ESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our Npac ChIP-seq results reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with p-TEFb, RNA Pol II Ser2 and Ser5. Depletion of Npac disrupts transcriptional elongation of pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for transcriptional elongation of pluripotency genes by recruiting of p-TEFb and interacting with RNA Pol II Ser2 and Ser5.

BackgroundBone morphogenetic protein type II receptor (BMPR2) gene mutation accounts for 80% of patients with heritable pulmonary artery hypertension (HPAH), and female mutation carriers have significantly higher penetrance rate than males. The inducement of HPAH penetrance and the mechanism of sex differential penetrance are still elusive. MethodsWe infected or transfected pulmonary artery smooth muscle cells (PASMCs) with shBMPR2 lentivirus or siBMPR2 to simulate the pathologic condition of BMPR2 heterozygous mutation and treated them with progesterone. The HPAH patient-derived induced pluripotent stem cells (iPSCs) were induced into vascular smooth muscle cells (VSMCs) to further verify the cellular phenotype. Wild-type flox+/- female mice and SM22-cre BMPR2 flox+/- female mice (CKO mice) were administered with 1-month progesterone, and their phenotype of PAH was evaluated by right heart catheterization and histopathological examination. ResultsProgesterone promoted the proliferation of PASMCs with BMPR2-knockdownby activating ERK pathway via progesterone receptor (PGR). Activated ERK not only upregulated the phosphorylation and elevation of cMYC, but also induced the transcription of endothelin (EDN1) by promoting the nuclear entry of c-JUN and combination on the its promoter region. Similar results were confirmed by iPSCs-VSMCs experiment. CKO mice developed PAH spontaneously and had increased expression of EDN1, which was further aggravated by exogenous progesterone. ConclusionsProgesterone might be an inducement of HPAH penetrance caused by BMPR2 mutation, accounting for sex differential penetrance.

ObjectiveUnwanted angiogenesis is involved in the progression of various malignant tumors and cardiovascular diseases, and the factors that regulate angiogenesis are potential therapeutic targets. We tested the hypothesis that DCBLD1 (Discoidin, CUB, and LCCL domain-containing protein 1) is a co-receptor of VEGFR-2 and modulates angiogenesis in endothelial cells(ECs). Approach and ResultsA carotid artery ligation model and retinal angiogenesis assay were used to study angiogenesis using globe knockout or EC-specific conditional DCBLD1 knockout mice in vivo. Immunoblotting, immunofluorescence staining, plasma-membrane subfraction isolation, Co-immunoprecipitation and mass-spectrum assay were performed to clarify the molecular mechanisms.Loss of DCBLD1 impaired VEGF response and inhibited VEGF-induced EC proliferation and migration. DCBLD1 deletion interfered with adult and developmental angiogenesis. Mechanistically, DCBLD1 bound to VEGFR-2 and regulated the formation of VEGFR-2 complex with negative regulators: protein tyrosine phosphatases, E3 ubiquitin ligases(Nedd4 and c-Cbl), and also DCBLD1 knockdown promoted lysosome-mediated VEGFR-2 degradation in ECs. ConclusionsThese findings demonstrated the essential role of endothelial DCBLD1 in regulating VEGF signaling and provided evidence that DCBLD1 promotes VEGF-induced angiogenesis by limiting the dephosphorylation, ubiquitination, and lysosome degradation after VEGFR-2 endocytosis. We proposed that endothelial DCBLD1 is a potential therapeutic target for ischemic cardiovascular diseases by the modulation of angiogenesis through regulating of the VEGFR-2 endocytosis.

Recent studies have revealed that tRNA modification is an important epigenetic mechanism involved in gene expression. Cytosolic thiouridylase (consisting of Ctu1 and Ctu2 subunit) are the enzyme complex which catalyze the thio-modification at the 34th wobble uridine of the anticodon of tRNAGlnUUG, tRNAGluUUC, and tRNALysUUU. Besides introducing a thiol group at the C2 positions, those tRNAs were commonly modified with a methoxycarbonylmethyl at the C5 positions by Elongator and ALKBH8. tRNA-U34 modification, particularly the Elongator and ALKBH8, has been demonstrated to be involved in disease and development, however, the biological functional level of CTU in vertebrates remains elusive. Here, we found that in zebrafish, CTU may be an important regulatory factor in development and erythroid differentiation. By using morpholino targeting and knocking down CTU1, we observed that the loss of CTU1 led to impaired zebrafish larval development and blood vessel formation. Single-cell sequencing analysis showed that erythroid cell differentiation in the CTU1 knockdown group was blocked at an early stage, while the wild-type group exhibited mature erythroid cells. These findings suggest that CTU1 is involved in regulating erythrocyte development. These findings provide new insights into the biological function of CTU1.

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.

The treatment of uterine fibroids and the development of new drugs depend on adeeper understanding of the developmental mechanisms of uterine fibroids. Here, the role of ESR1 and miR-17 on the uterine fibroids cell proliferation and apoptosis and their relationship were investigated in USMCs. Our results showed that ESR1 increased the proliferation of USMCs and inhibited their apoptosis. In addition, ESR1 could directly bind the promoter regionof TP53 and inhibit its expression. MiR-17 increased the apoptosis of USMCs and inhibited their proliferation via decreasing the level of ESR1 by targeting its 3UTR. Our research provides a new understanding of the development of uterine fibroids and provides a theoretical basis for the treatment of uterine fibroids.

Moyamoya disease (MMD) is a chronic cerebrovascular disorder characterized by progressive occlusion of the intracranial arteries, resulting in severe ischemic or hemorrhagic stroke. The main characteristic of the affected vessels in MMD is arterial intimal thickening. However, there are no in vitro or in vivo models that can mimic its vascular characteristics. Moreover, the mechanisms underlying the intimal thickening remain unexplained. Here, we generated vascular organoids by differentiating human induced pluripotent stem cell derived from the peripheral blood of MMD patients, thereby creating an organoid model reflecting both the genetic background and characteristics of the affected vessels. Through single-cell sequencing, we found the increased vascular smooth muscle cell (VSMC) proportion and its functional abnormalities in MMD organoids. Proteomics and RNA sequencing identified abnormal TUBA4A and TUBB4B overexpression in both the organoids and patient serum. The following in vitro experiments demonstrated that TUBA4A and TUBB4B promote the contractile-to-synthetic phenotypic switching, migration and proliferation in VSMC. Further experiments identified the GJA1-mediated PI3K/AKT/KLF4 pathway as a key regulating pathway of these phenotypic changes in VSMCs. Our findings demonstrate that the abnormal expression of TUBA4A and TUBB4B in VSMC might be a significant contributor to the intimal thickening in the affected vessels of MMD.

A coupled-clock system intrinsic to sinoatrial node (SAN) pacemaker cells that regulates the rate and rhythm of spontaneous action potential firing, is activated by Ca2+/calmodulin-stimulated Adenylyl Cyclase (AC) types 8 and 1. Our previous work in mice with cardiac specific overexpression of human AC8 gene (TGAC8) discovered that compared to its wild-type (WT) littermates, the heart rate (HR) of TGAC8 is elevated (by about 30%, 24 hours a day, 7days a week), and that the TGAC8 heart rhythm is markedly coherent, i.e., the HR Variability (HRV) lacks complexity, similar to that associated with aging or cardiac pathology. Reprogramming of molecular mechanisms, particularly SAN transcriptomic regulation that underlies the remarkable chronic shift in HR and HRV, however, has not been delineated. We conducted deep RNA sequencing (RNA-seq) in TGAC8 and WT SANs, using Mm10plus with human ADCY8 DNA sequence as the reference genome. Utilizing multiple bioinformatic techniques, we not only profiled the expression of marker genes related to SAN functions and AC-cAMP-PKA signaling, but also discovered negatively enriched hub pathways that differed in TGAC8 vs. WT, the top three being OXPHOS, ribosome, and cardiac muscle contraction. In contrast, signaling pathways related to inositol phosphate and its metabolism were positively enriched in TGAC8. Further, we identified two transcription regulators, KDM5A and PPARGC1A, that mediate effects of TGAC8 on ribosome and mitochondria. In summary, reprogrammed transcriptional regulation increases the HR of TGAC8 at the cost of impairment of other SAN cell functions, i.e., altered ribosome and inositol phosphate signaling and its crosstalk with mitochondria. Because these cell signaling alterations are associated with cardiac aging and age-associated CVDs, the TGAC8 mouse appears to be an ideal model in which to probe for potential therapeutic targets for cardiac aging and age-associated CVDs.

Hypertrophic scar (HS) is a prevalent yet unresolved wound healing complication characterized by persistent hyperactive and proliferative fibroblasts, leading to excessive extracellular matrix (ECM) synthesis and collagen contraction. Our previous studies have identified epidermal stem cells (ESCs) as critical for wound healing and HS remodeling, with its extracellular vesicles (EVs) playing a vital role. However, the specific mechanisms remain unclear. In this study, we first discovered that ESC-EVs could effectively induce the mesenchymal-epidermal transition (MET) of HS fibroblasts (HSFs) and inhibit their biological activity. Furthermore, by next-generation sequencing and multiplexed CRISPR/Cas9 system, we elucidated that this therapeutic effect is mediated by the miR-200 family (miR-200s) encapsulated in ESC-EVs, which targeted and inhibited ZEB1 and ZEB2 in HSFs. This vital role and mechanism have been thoroughly validated in both in vitro cell experiments and in vivo rat tail HS (RHS) models. These findings not only shed light on a previously unidentified mechanism of ESC-EVs for HS, but also provide potential novel targets and strategies for its precise treatment.

One striking feature of the large KRAB-containing zinc finger protein (KZFP) family is its rapid expansion and divergence since its formation about 400 million years ago. However, the evolutionary characteristics of KRAB domains, C2H2 zinc fingers and the full protein of KZFPs have not been fully analyzed. As for the drivers of the rapid evolution, its partly due to their coevolution with transposable elements (TEs). But their diverse functions besides inhibiting TEs suggest other reasons exist. Here we address these two issues by the systematic analysis of the divergence time and diversification pattern of KZFPs at three aspects and the functional analysis of the potential target genes besides TEs. We found that old-zinc-finger-containing KZFPs tend to have varied and disordered KRAB domains, indicating there are two ways of the evolution of KZFPs, including the variation of KRAB domains and the diversification of zinc fingers. Among them, the divergence of zinc fingers mainly contributes to the rapid evolution of KZFPs. Thus, we mainly focused on the functional requirements of the evolution of zinc fingers. Different from the classical regulation pattern of this family, we found and experimentally confirmed that KZFPs tend to bind to non-TE regions and can positively regulate target genes. Although most young genes tend to be with a low expression level, young-zinc-finger-containing KZFPs tend to be highly expressed in early embryonic development or early mesoderm differentiation, indicating their particular evolutionarily novel functional roles in these two processes. We further validated a young KZFP, ZNF611, can bind to non-TE region of STK38 gene and positively regulates its expression in ESCs. The emergence of new sequence in STK38 promoter may drive the evolution of zinc fingers in ZNF611. Finally, we proposed a two-way evolution model of KZFP family.

Understanding the molecular regulation of arterial and hemogenic specification during early embryonic vascular development is crucial for guiding vascular and hematopoietic regeneration. Accumulating evidence emphasizes the role of long non-coding RNAs (lncRNAs) in cell fate decision. However, the dynamic expression and the potential roles of lncRNAs in early vascular development are still unknown. Here, we first constructed a single-cell landscape of lncRNA expression based on the deeply sequenced tag-based single-cell transcriptome data of early embryonic vascular endothelial cells (VECs). We revealed the contribution of lncRNAs to VEC heterogeneity and identified 295 lncRNAs with specific expression in eight VEC populations. Furthermore, we identified a series of lncRNAs potentially involved in regulating the two waves of arterial specification and hemogenic specification. We uncovered a transient downregulation of H19 in the hemogenic endothelial population during endothelial-to-hematopoietic transition. Additionally, we constructed a transcription factor regulatory network composed of 287 regulons for early VEC development. We further revealed differential activation patterns of regulons and modules in the eight VEC populations, and predicted potential lncRNA-regulon regulatory network. Moreover, unsupervised analysis of the lncRNA expression profile revealed novel VEC subpopulations strongly associated with the maturation of VECs, suggesting the prominent roles of lncRNAs in endothelial maturation. In summary, our study fills the gap in understanding of lncRNA regulatory networks in early vascular development and provides insights into the fields of vascular and hematopoietic regeneration research.

Cardiac manifestations are commonly observed in COVID-19 patients and prominently contributed to overall mortality. Human myocardium could be infected by SARS-CoV-2, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection. However, molecular mechanisms of SARS-CoV-2 gene-induced injury and dysfunction of human CMs remain elusive. Here, we find overexpression of three SARS-CoV-2 coding genes, Nsp6, Nsp8 and M, could globally compromise transcriptome of hPSC-CMs. Integrated transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with hPSC-CMs of Nsp6, Nsp8 or M overexpression identified concordantly activated genes enriched into apoptosis and immune/inflammation responses, whereas reduced genes related to heart contraction and functions. Further, Nsp6, Nsp8 or M overexpression induce prominent apoptosis and electrical dysfunctions of hPSC-CMs. Global interactome analysis find Nsp6, Nsp8 and M all interact with ATPase subunits, leading to significantly reduced cellular ATP level of hPSC-CMs. Finally, we find two FDA-approved drugs, ivermectin and meclizine, could enhance the ATP level, and ameliorate cell death and dysfunctions of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Overall, we uncover the global detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, define the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explore the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities.

ObjectiveThis study explores the role of ZHX2 in vascular remodeling, specifically focusing on its effects on VSMC proliferation, migration, and neointima formation following vascular injury. Methods and Results: Data from both human atherosclerotic samples and a mouse carotid injury model indicated a decrease in ZHX2 levels. In vivo, ZHX2 overexpression reduced neointima formation in mice subjected to carotid artery ligation. In vitro, ZHX2 inhibited the proliferation and migration of primary VSMCs. Conversely, specific knockout of ZHX2 in SMCs in vivo or knockdown of ZHX2 in primary VSMCs had opposite effects. RNA- seq analysis revealed that ZHX2 overexpression significantly affected the expression of cell cycle-related genes. Using Chromatin Immunoprecipitation Sequencing (ChIP-seq) and luciferase reporter assays, we demonstrated that ZHX2 plays a crucial role in the transcriptional regulation of GADD45G, identifying GADD45G as the downstream target responsible for ZHX2-mediated effects. Conclusions: ZHX2 emerges as a key player in pathological vascular remodeling, suppressing VSMC proliferation and migration through its regulatory impact on GADD45G transcription and cell cycle-related gene expression.

Herbal medicine, especially Traditional Chinese medicine (TCM), is a valuable resource of natural products for drug discovery with obvious therapeutic effects. However, the mechanisms of action (MOAs) of herbal medicine are often unknown due to limited target information and the complexity of multiple ingredients and targets. This study developed a Herb-CMap algorithm to prioritize active ingredients and targets within herbal medicine by integrating transcriptomics- based gene perturbation data with a random walk algorithm. This methodology bridges the gap between gene perturbation of herbal medicine and its therapeutic target. Using the Suhuang antitussive capsule (Suhuang) for treating cough variant asthma (CVA) as a case study, we identify and experimentally verify that quercetin and luteolin directly interact with Il17a, Pik3cb, Pik3cd, Akt1, and Tnf. These interactions inhibit the IL-17 signaling pathway and inactivate PI3K, Akt, and NF- {kappa}B, preventing lung inflammation and treating CVA. Our findings demonstrate the potential of the Herb-CMap methodology to provide insights into the molecular MOAs of herbal medicine, thereby advancing drug discovery from herbal medicine.

In this study, we generated self-assembly cardiac organoids (COs) from human pluripotent stem cells by dual-phase modulation of Wnt/{beta}-catenin pathway, utilizing CHIR99021 and IWR-1-endo. The resulting COs exhibited a diverse array of cardiac-specific cell lineages, cardiac cavity-like structures and demonstrated the capacity of spontaneous beating and vascularization in vitro. We further employed these complex and functional COs to replicate conditions akin to human myocardial infarction and SARS-CoV-2 induced fibrosis. These models accurately captured the pathological characteristics of these diseases, in both in vitro and in vivo settings. In addition, we transplanted the COs into NOD SCID mice and observed that they survived and exhibited ongoing expansion in vivo. Impressively, over a span of 75-day transplantation, these COs not only established blood vessel-like structures but also integrated with the host mices vascular system. It is noteworthy that these COs developed to a size of approximately 8 mm in diameter, slightly surpassing the dimensions of the mouse heart. This innovative research highlighted the potential of our COs as a promising avenue for cardiovascular research and therapeutic exploration.