Cell Proliferation

Wound healing is a tightly orchestrated physiological process governed by dynamic cell-cell and cell-matrix interactions, yet how hypoxic microenvironments regulate migratory behavior in cells with latent lineage plasticity remains fully elucidated. Here, utilizing human embryonic kidney (HEK293T) and Madin-Darby Canine Kidney (MDCK) cells as a genetically tractable model, we investigate the cellular and molecular mechanisms driving hypoxia-accelerated collective wound repair. Time-lapse live-cell imaging and morphometric analyses reveal that hypoxic exposure significantly accelerates migration, shifts cell cycle dynamics toward the S/G2/M proliferative phases, and induces pronounced morphological spreading. Mechanistically, hypoxia induces a persistent, time-dependent downregulation of the desmosomal cadherin desmoglein-2 (DSG2), thereby weakening intercellular cohesion. Concurrently, the cell-matrix adhesion molecule integrin {beta}3 (ITGB3) exhibits a distinctive biphasic kinetic response--an initial sharp upregulation followed by a sustained decline-which serves to optimize focal adhesion traction and subsequent trailing-edge detachment. Transcriptomic profiling further corroborates these phenotypic transitions, demonstrating a global enrichment of gene networks associated with plasma-membrane adhesion organization, receptor activity, and ion homeostasis that independently mirrors the altered junctional dynamics and accelerated cellular responses. Collectively, our findings uncover a novel cooperative mechanism by which hypoxic stress coordinates cell-cell and cell-matrix adhesion remodeling to facilitate efficient tissue repair, highlighting the valuable utility of plastic cellular models in decoding microenvironmental stress responses.

Embryo polarization is critical for the first cell fate segregation. While mechanisms underlying its initiation have been described, the intrinsic signaling pathways that regulate this process remain poorly understood. Here, we show that mouse embryonic stem cells, when aggregated under defined conditions, recapitulate the first lineage segregation to generate trophectoderm (TE)-like cell populations and undergo self-organized morphogenesis into blastocyst-like structures. In the blastoid-forming medium, we identify CHIR99021 is essential for the generation of blastoids from both ESCs and totipotent-like cells. CHIR99021 promotes cell polarization and TE differentiation by activating the WNT/{beta}-catenin pathway and upregulating associated genes. Consistent with this, genetic ablation of {beta}-catenin abolished the cell polarization and disrupted blastoid formation from ESCs, a defect that was restored by {beta}-catenin overexpression. Moreover, {beta}-catenin depletion compromised cell polarization in natural embryos. Collectively, this study establishes the Wnt/{beta}-catenin as a critical regulator initiating polarization in vitro and in mouse early embryo development.

BackgroundCalcific aortic valve disease (CAVD) is the most prevalent valvular heart disease, yet there is no effective pharmacological therapy to halt or reverse its progression. Focal adhesions (FAs) are dynamic structures that connect cells to the extracellular matrix (ECM), serving not only as mechanical anchors but also as critical signalling hubs that regulate cell adhesion, spreading, migration, differentiation, and mechanotransduction. However, their specific role in CAVD pathogenesis remains largely unknown. MethodsTo identify upregulated hub genes, an integrated analysis of proteomics and RNA sequencing was performed on calcified aortic valves. To investigate functional roles in vitro, we utilized genetic knockdown and overexpression of VASP in valvular interstitial cells (VICs), followed by phenotypic evaluations of osteogenic differentiation, calcification, and elastin (ELN) secretion. Downstream mechanisms were explored using RNA-seq in VASP-overexpressing VICs, alongside pharmacological inhibition of the FA pathway. Finally, protein-protein interaction assays were conducted to map and analyze the physical binding between VASP and the structural domains of FBLIM1. ResultsIntegrated omics analysis identified VASP as a significantly upregulated gene in human calcified aortic valve tissues. Functionally, VASP knockdown inhibited, whereas its overexpression promoted, the osteogenic differentiation of VICs. RNA-seq revealed that VASP overexpression activated the FA pathway, and the pharmacological blockade of this pathway successfully suppressed calcification both in vitro and vivo. Mechanistically, we demonstrated a direct physical interaction between VASP and the third LIM zinc-binding domain of FBLIM1, and knockdown of FBLIM1 effectively reversed the pro-calcific effects of VASP. Finally, VASP overexpression was found to promote the secretion of ELN, which subsequently contributed to the calcification process. ConclusionOur study reveals that the interaction between VASP and FBLIM1 drives CAVD progression by activating the FA pathway, which subsequently leads to excessive ELN secretion. These findings reveal a novel mechanistic pathway and may provide a potential therapeutic target for CAVD intervention. What are the Clinical Implications?We have identified the VASP-FBLIM1-FA-ELN axis as a novel molecular pathway involved in CAVD pathogenesis, greatly enriching our understanding of the complex process of aortic valve calcification. This pathway intricately links cytoskeletal dynamics, cell adhesion signalling, and ECM remodelling, providing a new theoretical framework for pathophysiological research in CAVD. In clinical practice, VASP and its downstream pathway components, particularly FBLIM1 and the FA pathway, may serve as potential biomarkers and therapeutic targets for the early diagnosis and treatment of CAVD. For example, the FA pathway inhibitors PF-573228 and Y15 demonstrated significant anti-calcification effects both in vitro and in vivo, suggesting that targeting this pathway may offer a new nonsurgical intervention strategy for patients with CAVD.

BackgroundEndothelial cell (EC) injury induced by disturbed flow drives neointimal hyperplasia in arteriovenous fistulas (AVFs), where CD34+ cell-mediated repair may be involved. PIEZO1 and VEGFR2 are important mechanosensors with critical role in maintaining endothelial function. However, whether PIEZO1 interacts with VEGFR2 during CD34+ cell differentiation to orchestrate the vascular repair remains unknown. MethodsAVF model was established in several mouse strains. Single cell RNA sequencing was performed for human and mouse samples. Cd34-CreERT2; R26-tdTomato; Piezo1flox/flox mice were used to investigate the effect of Piezo1 deletion on endothelial repair in AVFs. CD34-high human umbilical vein ECs (CD34high HUVECs) was sorted and exposed to different flow patterns to determine the role of shear stress in CD34high cell differentiation. Co-immunoprecipitation, proximal ligation assay and complementary approaches were performed to delineate mechanotransduction initiated by PIEZO1-VEGFR2 interaction. ResultsSingle cell RNA sequencing and immunostaining showed abundant CD34high cells in the vessel wall of AVFs in humans and animal models. Exposure of CD34high HUVECs to different flow patterns showed that laminar shear stress downregulated CD34 while upregulating VE-cadherin and claudin-5 expression. In contrast, oscillatory flow produced the opposite effects, indicating impaired endothelial maturation. PIEZO1 knockdown in CD34high HUVECs attenuated shear stress-induced endothelial marker expression. In Cd34 conditional Piezo1 knockout mouse model of AVF, we observed decreased number of CD34-derived cells, more compact cellular arrangement, and attenuated neointimal hyperplasia. Mechanistically, we found PIEZO1 interacts with VEGFR2, thereby mediating the distinct effects of laminar and oscillatory shear stress on AKT-FoxO1 axis, which critically regulates endothelial marker expression. Furthermore, pharmacological activation of AKT signaling in AVF mouse model enhanced CD34+ cell-mediated endothelial repair and attenuated neointimal hyperplasia. ConclusionPIEZO1-VEGFR2 complex-mediated mechanotransduction plays a key role in regulating CD34+ cell-derived endothelial repair in AVFs via AKT-FoxO1 axis. AKT activation enhances endothelial maturation, thereby attenuating neointimal hyperplasia in AVFs. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn arteriovenous fistulas (AVFs), abnormal shear stress induces endothelial cell injury, and the resulting neointimal hyperplasia is a major cause of anastomotic stenosis. C_LIO_LICD34 cells actively participate in vascular endothelial repair. C_LIO_LIPIEZO1 is a mechanoreceptor mediating endothelial sensing of hemodynamic shear stress, contributing to the maintenance of atheroprotective endothelial phenotype under laminar shear stress, whereas its activation induces pro-inflammatory effects under disturbed shear stress. C_LI What New Information Does This Article Contribute?O_LICD34 cells participate in repairing endothelial injury induced by abnormal shear stress in AVFs. PIEZO1 knockout in CD34+ cells improve endothelial repair and attenuates neointimal hyperplasia in AVF. C_LIO_LILaminar shear stress induces CD34 downregulation and upregulates VE-cadherin and claudin-5 expression in CD34-high human umbilical vein endothelial cells, whereas oscillatory shear stress upregulates CD34 expression and suppresses VE-cadherin and claudin-5 expression. C_LIO_LIMechano-stimuli lead to PIEZO1-VEGFR2 complex formation regulating CD34 cell-mediated endothelial repair through the downstream AKT-FoxO1 axis. C_LI Abnormal hemodynamic shear stress-induced endothelial injury initiates neointimal hyperplasia in AVFs. The present study identifies PIEZO1 as a key mechanosensor that regulates CD34+ cell-derived endothelial repair in response to distinct blood flow patterns. PIEZO1 promotes CD34+ cell differentiation into mature ECs for endothelial repair under laminar shear stress, whereas it disrupts the differentiation of CD34+ cells into mature endothelium under oscillatory shear stress. Mechanistically, a novel shear stress-sensing complex comprising PIEZO1 and VEGFR2 was identified in regulating flow-induced differentiation of CD34+ cells into mature ECs via the AKT-FoxO1 signaling axis, thereby controlling the expression of endothelial maturation markers VE-Cadherin and Claudin-5. These findings define a novel PIEZO1-VEGFR2 mechanotransduction axis in CD34+ cell-mediated endothelial repair and support AKT pathway activation as a potential therapeutic strategy against neointimal hyperplasia in AVFs.

BACKGROUNDEndothelial cell senescence induces endothelial dysfunction, thereby contributing to atherosclerosis progression. Circular RNAs (circRNAs) play diverse roles in multiple physiological and pathological processes. N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic RNAs and dynamically regulates RNA fate and function. However, the functions and therapeutic potential of m6A-modified circRNAs in endothelial cell senescence remain unknown. METHODSm6A-modified circRNAs associated with endothelial cell senescence were screened by circRNA expression and m6A-circRNA microarray profiling of endothelial cells and mouse aortic intima. circEZH2 expression was validated in endothelial cells, vascular tissues, and human atherosclerotic plaques by RT-qPCR, and RNA fluorescence in situ hybridization. The role of circEZH2 in endothelial senescence and atherosclerosis was assessed in vitro and in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, co-immunoprecipitation, ubiquitination assays, and rescue experiments were used to define the underlying mechanism. RESULTSWe identified A novel m6A-modified circRNA, circEZH2, that was downregulated in the aged aortic intima and advanced plaques. CircEZH2 was stabilized by m6A reader IGF2BP2. Endothelial cell-specific overexpression of circEZH2 delayed senescence and suppressed atherosclerosis progression. At the cellular level, circEZH2 overexpression delayed senescence, decreased p53/p21 levels and increased angiogenic activity of endothelial cells, while circEZH2 knockdown exhibited the opposite effect. Mechanistically, circEZH2 functions as a scaffold to promote USP37-mediated deubiquitination, thereby stabilizing ZNF326. Moreover, endothelial cell-specific knockdown of ZNF326 counteracts the anti-senescent and anti-atherosclerotic effects mediated by circEZH2 overexpression. CONCLUSIONSIn summary, the present study identifies circEZH2 as a novel suppressor of endothelial cell senescence, highlighting its potential as a therapeutic target for age-related atherosclerosis. GRAPHIC ABSTRACTA graphic abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/730538v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1bce806org.highwire.dtl.DTLVardef@124ff87org.highwire.dtl.DTLVardef@40911org.highwire.dtl.DTLVardef@ef3d26_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Schematic model of m6A-modified circEZH2 regulation in endothelial cell senescence and atherosclerosis. In young endothelial cells, IGF2BP2 is highly expressed and recognizes m6A-modified circEZH2, thereby maintaining its RNA stability. CircEZH2 stabilizes ZNF326 protein through USP37-mediated deubiquitination, which leads to suppression of p21 and p53, delays endothelial cell senescence, ameliorates endothelial dysfunction, and ultimately suppresses the progression of atherosclerosis. C_FIG What Are the Clinical Implications?This study identifies circEZH2 as a novel m6A-modified circular RNA that is reduced in the aged aortic intima and in endothelial cells within advanced atherosclerotic plaques. Endothelial circEZH2 overexpression delays endothelial cell senescence, preserves endothelial function, and suppresses atherosclerotic lesion formation, supporting an important role for circEZH2 in vascular aging-associated atherosclerosis. Mechanistically, circEZH2 acts as a scaffold to enhance USP37-mediated deubiquitination and stabilization of ZNF326, while endothelial ZNF326 knockdown counteracts the anti-senescent and anti-atherosclerotic effects of circEZH2. These findings reveal the circEZH2-ZNF326 axis as a previously unrecognized mechanism regulating endothelial senescence and atherosclerosis progression. Our work supports the potential of circEZH2-based gene therapy as a novel therapeutic approach for atherosclerosis.

The tumor microenvironment (TME) plays a critical role in cancer progression and therapeutic response, with cancer-associated fibroblasts (CAFs) being a key stromal component. Conventional tumor organoid models lack TME elements, and existing co-culture systems have limitations in recapitulating dynamic, multidimensional interactions. Here, we developed a novel dynamic co-culture chip (BAC) to better model the TME and investigate CAF-tumor cell crosstalk. Using this platform, we established a non-contact co-culture model of patient-derived colorectal cancer cells and CAFs. The resulting model closely recapitulated the morphological and molecular characteristics of the original patient tumors. Compared with tumor organoids cultured alone, the co-culture system exhibited significantly higher resistance to the clinically common chemotherapeutics 5-fluorouracil and oxaliplatin. Moreover, the presence of CAFs promoted tumor recurrence. Notably, drug responses in the co-culture model showed superior concordance with clinical outcomes relative to both organoid-only and animal models. Transcriptomic profiling under different culture conditions provided further insights into the mechanisms driving CAF-mediated interactions. These findings demonstrate that the BAC-based tumor organoid-CAF co-culture model serves as a more accurate platform for predicting drug responses, investigating TME-dependent mechanisms, and guiding personalized cancer therapy.

Multicellular spheroids better recapitulate native cardiac tissue than two-dimensional systems, preserving cell-cell and cell-matrix interactions and relevant signalling. However, analytical tools for extracting quantitative data from these complex models remain limited. Here, we present an optimised holotomography (HT) workflow for fixed spheroids, enabling label-free quantification of protein concentration and dry mass across conditions. Using a hypoxia-reperfusion injury model to mimic myocardial infarction, HT measurements reveal a statistically significant reduction in the protein concentration of cardioids, reflecting impaired structural integrity and declining viability, subtle changes often missed by conventional approaches. These findings establish HT as a robust, scalable method for quantitative analysis of 3D cardiac models, with direct relevance for disease modelling and preclinical research.

HighlightsO_LIIdentified hundreds of potential chromatin-associated circRNAs in HEK293 cells, H9, and HeLa cells C_LIO_LIThe first report suggesting global interaction of circular RNAs with chromatin C_LIO_LIChromatin-associated circular RNAs interact with various RBPs involved in RNA splicing or processing C_LI Circular RNAs (circRNAs) have emerged as novel regulators of gene expression by interacting with various proteins and RNAs in a spatiotemporal manner. CircRNAs localized in the cytoplasm regulate mRNA translation or stability by binding to microRNAs and RNA-binding proteins (RBPs), while circRNAs in the nucleus regulate transcription and pre-mRNA splicing by associating with transcription factors and splicing factors. In this study, we sought to explore the interaction between circRNAs and chromatin. Analyzing published RNA-seq data from chromatin fractions identified hundreds of chromatin-associated circRNAs (cacRNAs) in various human cells. We validated the enrichment of a subset of circRNAs in the chromatin fraction and established the direct interaction of circDYNC1H1 and circKIF2C with chromatin in HEK293T cells. Furthermore, cacRNAs were found to interact with RBPs. Together, our research demonstrates the global association of hundreds of circRNAs with chromatin and expands our understanding of novel functional aspects of the circRNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/739476v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1fc8831org.highwire.dtl.DTLVardef@516cdcorg.highwire.dtl.DTLVardef@1c20395org.highwire.dtl.DTLVardef@79313a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

ObjectiveTo investigate and clarify the role of Ginsenoside Ro (GRo) in diabetic cardiomyopathy (DiaCM) and to elucidate the molecular mechanism by which GRo ameliorates DiaCM. Methods[circled1] The construct of type 2 diabetic mouse model. The bought C57BL/6 male mice were housed in a specific pathogen-free (SPF) animal facility and randomly divided into control, STZ (model), STZ + GRo, and control+GRo groups. The STZ (model) and STZ + GRo groups were fed a high-fat and high-glucose diet combined with intraperitoneal injection of streptozotocin (STZ). The control and control + GRo groups were fed a normal diet, while the control + GRo and STZ + GRo groups were treated with GRo via oral gavage. Then, all groups were evaluated for cardiac function and structure by small animal echocardiography and histological staining including hematoxylin and eosin (HE) and Massons trichrome staining to screen and confirm diabetic cardiomyopathy in mice. Finally, immunofluorescence staining of cilia in mouse heart tissue was performed to determine whether GRo inhibits abnormal ciliary growth. [circled2] The construct of cell models. First, the CCK-8 (Cell Counting Kit-8) assay was used to separately evaluate the cytotoxicity of GRo and the combination of TGF-{beta}1 and PA in myocardial fibroblasts and cardiomyocytes. Subsequently, mouse myocardial fibroblast lines (MCFs) were treated with transforming growth factor-beta 1 (TGF-{beta}1), and H9c2 cardiomyocytes were treated with palmitic acid (PA). Both cell types then received the GRo treatment. [circled3] Molecular and cellular testing. Firstly, we measured serum levels of cardiac injury markers (CK-MB, MYO, and TNNI3), glutathione (GSH), and malondialdehyde (MDA). Secondly, we examined the expression of myocardial fibrosis-related genes (Col1a1, etc.), myocardial hypertrophy markers (Nppa, etc.), cilia-specific genes (Pkd1, etc.), and oxidative stress-related genes (Nrf2, etc.) in both animal and cell samples by Western blotting and RT-qPCR. Finally, we used immunofluorescence staining of myocardial fibroblasts to detect cilia length and phalloidin staining of cardiomyocytes to measure their cross-sectional area. [circled4] The correlation mechanism. Firstly, the cilia-specific inhibitory drug HIP-4 was used to disrupt cilia homeostasis by inhibiting cilia growth. Secondly, small activating RNA (saRNA) was used to upregulate the Pkd1 gene to verify whether GRo exerts its anti-fibrotic effects through the inhibition of PC1. Results[circled1] Animal level. A diabetic cardiomyopathy mouse model was successfully established by combining STZ injection with a high-fat and high-glucose diet, and treatment with GRo significantly ameliorated the associated symptoms. [circled2] Cellular level. We successfully established a myocardial fibrosis model by treating myocardial fibroblasts with TGF-{beta}1, and a myocardial hypertrophy model by treating cardiomyocytes with PA. Immunofluorescence staining demonstrated that GRo significantly decreased cilia length in the fibrosis model, while phalloidin staining showed that GRo significantly attenuated the increase in cardiomyocyte cross-sectional area. [circled3] Molecular level. Compared with the model group, GRo treatment significantly reduced serum levels of cardiac injury markers (CK-MB, MYO and TNNI3), glutathione (GSH) and malondialdehyde (MDA). Western blotting and RT-qPCR analyses of both animal and cell samples revealed that GRo markedly alleviated indicators of myocardial fibrosis and hypertrophy, while also suppressing cilia-specific genes and oxidative stress-related genes. Overall, GRo significantly ameliorated the markers associated with myocardial fibrosis and hypertrophy, and inhibited cilia-specific protein expression as well as oxidative stress parameters. [circled4] The correlation mechanism. The cilia-specific drug hedgehog pathway inhibitor 4 (HPI-4) was used to revealed that cilia homeostasis is closely linked to myocardial fibrosis and shortened cilia inhibit the fibrosis progression. Furthermore, upregulation of the Pkd1 gene by small activating RNA demonstrated that PC1 overexpression abrogates the therapeutic effect of GRo. Finally, GRo can alleviate DiaCM.

Sustained trophic factor delivery via Encapsulated Cell Technology (ECT) is a powerful new class of therapeutics with broad potential for targeted treatment. Intravitreal delivery of ciliary neurotrophic factor (CNTF) via the ECT, NT-501, is a first-in-class therapy that slows the progression of macular telangiectasia type 2 (MacTel). Despite its clinical efficacy, key questions remain regarding its mechanism of action, including whether other implant-derived factors contribute to therapeutic benefit and how optimal dosing should be determined. Resolving these issues is critical for optimizing NT-501 in MacTel and guiding the development of ECT-based therapies for other diseases. We evaluated the biological activity of implant-derived cytokines on retinal tissue, using long-term NT-501 intravitreal implants in rabbits alongside human retinal organoid (hRO) models treated with NT-501-conditioned medium (NT-501-CM). Then, using a MacTel-specific photoreceptor degeneration model in hROs, we showed NT-501-CM significantly reduced photoreceptor cell death, and this protective effect was abolished by either CNTF-neutralizing antibodies or JAK inhibitor. We also established a therapeutic dose-response relationship linking NT-501-derived CNTF levels to JAK/STAT3 activation and photoreceptor protection. These findings directly connect ECT-derived CNTF exposure with JAK/STAT3-mediated photoreceptor protection in human retinal tissue and suggest an optimal concentration range for efficacy.

Microplastics are pervasive environmental pollutants increasingly implicated in adverse human health effects, with emerging evidence linking MPLs exposure to elevated cardiovascular risk, including atherosclerosis. However, their specific mechanisms of action remain unknown. Human aortic endothelial cells (HAECs), located in the innermost layer of blood vessels, play a crucial role in maintaining vascular homeostasis and the development of atherosclerosis. This study demonstrates that polystyrene microplastics (80 nm MPLs) can enter HAECs through multiple pathways, including macropinocytosis, clathrin-mediated endocytosis, and caveolin-mediated endocytosis, and co-localize with mitochondria and lysosomes. MPLs exposure resulted in coordinated transcriptional, epitranscriptomic, and metabolomic reprogramming in HAECs, characterized by disruption of mitochondrial genes and an inflammatory response with activation of TNF-a; and NF-kB signaling. Integrative analysis revealed remodeling of the epitranscriptomic profile, demonstrated by an increase in 1-methyladenosine (m1A) modification along with reciprocal regulation (TRMT61A upregulation and ALKBH3 suppression) of its transcriptomic machinery, alongside other enzymes associated with 3-methylcytidine (m3C), pseudouridine (Y), 5-methylcytidine (m5C), and 7-methylguanosine (m7G) pathways. By comparing transcriptomic data from MPLs-treated HAECs with those of human atherosclerotic plaques, several common dysregulated pathways were identified, particularly those related to vascular physiological regulation and cell signaling. Metabolomic profiling further revealed significant remodeling of lipid metabolic networks associated with oxidative stress and inflammatory signaling. In summary, this study reveals that HAECs can internalize MPLs, leading to multiple disturbances in the transcriptome, epigenome, and metabolic networks, suggesting that MPLs exposure may pose a potential hazard to human cardiovascular health.

ObjectiveIn recent years, the number of cats kept as companion animals has increased, leading to a growing demand for veterinary care. Although some histone deacetylase (HDAC) inhibitors are promising for the treatment of human cancers and neurological diseases, comprehensive systematic research on HDAC inhibitors in domestic cats remains insufficient. Therefore, this study aimed to investigate the effects of HDAC inhibitors on the transcriptome of feline cells. MethodsTwo types of cells derived from domestic cats, Crandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived), were treated with four HDAC inhibitors (panobinostat, trichostatin A, valproic acid, and vorinostat) for 24 h. Transcriptomic changes after treatment were examined using RNA sequencing. ResultsHDAC inhibitor treatment upregulated the expression of intercellular chemical interactions and signal transduction-related genes, similar to observations in human cells. Although HDAC inhibitors did not suppress the expression of cell cycle-related genes in CRFK cells, as observed in human cells, the inhibitors downregulated the expression of organogenesis-related genes. Consistent with observations in human cells, HDAC inhibitors suppressed the expression of cell cycle- and cancer-related genes in PG-4 cells. Importantly, valproic acid, which is thought to be more effective for neurological diseases than for cancer, suppressed the expression of more cancer-related genes in PG-4 cells than the other three HDAC inhibitors. Conclusion and relevanceOur findings revealed that the responses of cells derived from feline organs to various HDAC inhibitors varied considerably depending on the organ of origin and species. Since few studies, including human studies, have comprehensively compared transcriptomic responses to multiple HDAC inhibitor classes across multiple cell types, the results of this study provide a foundation for future research on the treatment and prevention of cancer and neurological diseases in domestic cats and other mammals.

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

Background: Epigenetics, the study of reversible changes in gene expression without altering the underlying DNA sequence, is increasingly applied in medical, commercial, and policy contexts. Yet, little is known about how this emerging science is communicated to the public. The purpose of this study was to examine communication strategies, sources, and modalities in epigenetic-related videos on YouTube- the most accessed platform for informal science education. Methods: We conducted a mixed-methods content analysis of 294 YouTube videos on epigenetics by conducting a keyword-based search on October 17, 2023. Video transcripts and meta-data were coded using a codebook developed both deductively and inductively. Qualitative analysis examined how communication strategies were used within videos and identified emergent themes (RQ1). Quantitative analyses examined the frequency of video and channel characteristics (RQ2), and presentation modalities (RQ3). Results: Findings reveal poor alignment with science communication best practices (RQ1): over 92% of videos failed to acknowledge scientific uncertainty, the comprehensibility level exceeded the recommended 8th-grade level (e.g., average readability grade 10.7), and professional research organizations were notably absent. Narrators were mostly male (56.7%) and white-presenting (73.7%) (RQ2). The majority of the videos used multi-modal strategies (e.g., visual texts mixed with animation and voice-over narration) to communicate epigenetic information (RQ3). Conclusion: Findings highlight the need for professional research organizations to be more proactive in public epigenetic communication efforts. Increasing narrator demographic diversity could broaden audience reach. Evidence-based communication tools are needed for health or science communicators discussing epigenetics on social media.

Platelets are increasingly recognized as active regulators of tissue repair and liver regeneration beyond their classical roles in hemostasis and thrombosis. Loss of terminal sialic acid from platelet surface glycoproteins, a process known as desialylation, occurs during platelet aging or activation and has been linked to platelet clearance via the asialoglycoprotein receptor (ASGPR) on hepatocytes. However, the mechanisms by which desialylated platelets (D-plts) directly stimulate hepatocyte proliferation remain poorly understood. This study aimed to elucidate the proliferative effects of D-plts on hepatocytes and to identify the underlying signaling mechanisms. D-plts were generated and co-cultured with hepatocyte models exhibiting low or absent levels of asialoglycoprotein receptor 1 (ASGPR1) expression, including HepG2 cells, HuH-7 cells, and human chemically induced liver progenitors. Hepatocyte proliferation was assessed, and the roles of platelet-derived factors and downstream signaling pathways were investigated. Co-culture with D-plts significantly increased hepatocyte proliferation in all three cell models compared with the corresponding controls. Moreover, supernatants derived from stimulated D-plts also significantly enhanced hepatocyte proliferation, suggesting that soluble platelet-derived factors contribute to this effect. Mechanistically, the proliferative effects were mediated predominantly through the ERK1/2 signaling pathway rather than the JAK-STAT pathway in both hepatocytes co-cultured with D-plts and those treated with D-plt-derived supernatants. In conclusion, our findings demonstrate that D-plts directly promote hepatocyte proliferation through an ASGPR-independent pathway, in which ERK1/2 signaling plays a central role. These results highlight a novel mechanism through which platelet desialylation may contribute to liver regeneration. Graphical Abstract(A) Desialylated platelets are readily activated and release increased amounts of EGF, promoting hepatocyte proliferation via the EGF-ERK signaling pathway. (B) Normal platelets show lower reactivity and reduced EGF release than desialylated platelets, resulting in weaker hepatocyte proliferation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/740293v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1cbe93borg.highwire.dtl.DTLVardef@3d57c7org.highwire.dtl.DTLVardef@14dd63forg.highwire.dtl.DTLVardef@12cf508_HPS_FORMAT_FIGEXP M_FIG C_FIG

Subarachnoid hemorrhage (SAH) resulted from intracranial aneurysm (IA) rupture is an especially severe form of stroke. Endothelial dysfunction represents the initiating event of IA pathogenesis. Understanding the role of endothelial cells (ECs) underlying formation of IAs is helpful to seek for pharmaceutical treatment strategy. Based on single-cell RNA sequencing, proteomics, and metabolic analysis, we discovered a change in cell population in IA samples, majorly in ECs and macrophages (MPs). Abnormal ECs exhibit senescence and death in IA samples, which is absent in the control arterial samples. Cross-analysis of multi-omics revealed that CALM1, a calcium detector involved in mechanotransduction, is downregulated in the abnormal ECs. CALM1 knockdown leads to senescence and inhibits the proliferation and maturation of ECs under turbulent flow. Through high-throughput virtual screening, this work identified compound ZC04329651 as a potent CALM1 activator in maintaining the stability of endothelial cell junctions and attenuating cellular senescence. Thus, our findings showed compound ZC04329651 up-regulate the expression of CALM1 to restore ECs, which maybe a promising pharmacological treatment strategy for IAs.

Human iPSC-derived brain organoids are revolutionizing tools to study layers biology, synergize disease modeling, and accelerate therapeutic discoveries that overcome obstacles in monolayer cell culture or animal models. The neurovascular unit including vasculature and microglia is critical for brain development, maintenance of synaptic plasticity and neural activity, and the high metabolic demands of long-term culture. We present a methodology to incorporate these important components during organoid generation and discuss potential approach, aiming consistent production of vascularized organoids for longitudinal study. We also demonstrate that this vascularized organoid is a versatile platform to model brain cancer and traumatic brain injury.

During development, endothelial cells (ECs) migrate into the brain and acquire blood-brain barrier (BBB) properties such as tight junctions, limited transcellular transport, and high electrical resistance. Although key signaling pathways that are active in vivo have been identified, factors critical in inducing brain EC differentiation in vitro remain unclear. Here, we describe conditions that promote brain EC-like gene expression in human pluripotent stem cell (hiPSC)-derived ECs. Activation of Wnt/{beta}-catenin signaling upregulates the brain EC marker GLUT1 (SLC2A1) while suppressing the peripheral EC marker PLVAP. Simultaneously, stimulation of STAT3 by CNTF together with TGF-{beta} inhibition increases CLDN5 expression. We further found that hiPSC-derived ECs secrete high levels of angiopoietin-2 (ANGPT2) and that razuprotafib (AKB-9778), a PTPRB (VE-PTP) inhibitor, inhibits ANGPT2 and improves monolayer integrity. These results suggest that combinatorial modulation of specific signaling pathways stimulates the differentiation of human brain ECs in vitro.

BackgroundPathological intraplaque neovascularization, vascular leakage, and fibrous cap thinning contribute to vulnerable atherosclerotic plaque rupture. Platelet- derived growth factor-BB (PDGF-BB) has been shown to promote pericyte recruitment, thereby stabilizing the microvascular structure, and to induce phenotypic modulation of vascular smooth muscle cells (VSMCs), which enhances fibrous cap thickness and reinforces plaque stability. Nevertheless, systemic protein delivery is limited by rapid clearance and potential off-target effects. MethodsWe developed PDGF-BB mRNA-loaded lipid nanoparticle-poly(lactic-co- glycolic acid) nanobubble complexes (LNPmRNA@PLGA) and used low-intensity focused ultrasound (LIFU) to enhance plaque-targeted delivery. Cellular uptake, PDGF- BB expression, vascular mural-cell responses, plaque histology, hemodynamics, and proteomic changes were evaluated in vitro and in ApoE-/-Fbn1C1041G+/- mice. ResultsLIFU enhanced nanocomplex uptake and PDGF-BB expression, promoted vascular smooth muscle cell proliferation, migration, and phenotypic switching, and increased pericyte coverage. In vivo, LIFU plus LNPmRNA@PLGA reduced the plaque vulnerability index by 78.2% and the neovascularization area by 67.3% compared with controls, while increasing collagen deposition and improving carotid hemodynamics. ConclusionsLIFU-responsive delivery of PDGF-BB mRNA stabilized vulnerable plaques by promoting neovessel maturation and strengthening the fibrous cap. This strategy provides a spatially controlled framework for therapeutic remodeling of high-risk atherosclerotic plaques. Research Perspective What New Question Does This Study Raise?O_LICan spatially controlled PDGF-BB mRNA delivery simultaneously mature intraplaque neovessels and reinforce the fibrous cap without the systemic effects associated with recombinant PDGF-BB? C_LI What Question Should Be Addressed Next?O_LIFuture studies should define the therapeutic window, durability, and long-term safety of LIFU-triggered PDGF-BB mRNA delivery in large-animal models that more closely reproduce human plaque rupture. C_LI

BackgroundMetabolic reprogramming is a common occurrence in tumor cells, where enhanced glycolysis promotes cell growth, invasion and migration. NF1 is tumor suppressor gene that downregulates the encoded neurofibromin protein. However, the effects of NF1 on energy metabolism and epithelial-mesenchymal transition (EMT) in glioblastoma multiforme (GBM), as well as the underlying molecular mechanisms, remain unclear. MethodsCRISPR/Cas9 gene editing technology was employed to construct GBM cell lines with NF1 gene mutations. Metabolomics was utilized to examine the impact of NF1 on metabolic remodeling in GBM. The Seahorse XF24 extracellular flux analyzer was used to detect the effect of NF1 knockdown on glycolysis and mitochondrial oxidative phosphorylation in GBM cells. Wound healing assay and Transwell chamber assay were utilized to detect the effect of NF1 on GBM cell invasion. Orthotopic tumor model in nude mice was established to explore the role of NF1 in vivo. In addition, Co-IP, western blotting, and immunofluorescence were used to explore the changes of key enzymes in glycolysis and mitochondrial oxidative phosphorylation and the relationship between NF1 and MFN1. ResultsThe expression of NF1 is decreased in glioma tissues and is significantly correlated with patient prognosis. NF1 knockdown may promote the invasion, migration, and EMT of GBM cells. At the same time, the activation of the AKT/mTOR signaling pathway promotes aerobic glycolysis in GBM cells, promotes mitochondrial division through targeted regulation of MFN1, and inhibits mitochondrial oxidative phosphorylation. NF1 deficiency promotes EMT in GBM cells by enhancing aerobic glycolysis and mitochondrial division. ConclusionNF1 deficiency promotes GBM glycolysis by activating the AKT/mTOR signaling pathway and inhibits the mitochondrial oxidative phosphorylation by regulating MFN1; NF1 deletion promotes GBM EMT by remodeling the pattern of energy metabolism.