Intraocular vasculature formation precedes extraocular vasculature penetration of mouse eyes.

Abstract Introduction/Objective Testicular hemangiomas are rare neoplasms. Less than 60 such cases are reported worldwide, with majority of patients are younger than 20 years. All of the reported testicular hemangiomas are cavernous, capillary, epithelioid and anastomosing subtypes with exception of three reported lobular capillary hemangiomas and none had associated hydrocele. We present the first case of testicular lobular capillary hemangioma with associated hydrocele in an elderly patient. Methods/Case Report A 69-year-old man presented with a three-month history of painless right testicular mass. Scrotal ultrasonography demonstrated a hydrocele and 2-cm isoechoic, vascularized, intratesticular mass in the right testis. His serum tumor markers were normal. A right radical orchiectomy was performed as malignancy could not be excluded. A 1.9-cm intratesticular well-demarcated mass with hydrocele was identified grossly. Microscopically, the mass demonstrated a lobulated proliferation of benign endothelial cells, forming variably solid and compressed capillaries, and accentuating around larger vessels. These neoplastic cells were positive for CD31, CD34, and FLI-1 and negative for cytokeratin, calretinin, inhibin, and CD30, confirming endothelial cell differentiation. An adjacent hydrocele was also confirmed microscopically. Results NA Conclusion This unique and interesting case could help identify similar patients where a partial orchiectomy with a confirmatory frozen section might be considered instead of a radical orchiectomy.

Pulmonary Arterial Hypertension (PAH) is a leading cause of cardiovascular-related mortality worldwide. The emergence of single-cell RNA sequencing (scRNA-seq) has enhanced the ability to dissect cellular heterogeneity in PAH at a granular level. Transcriptome-wide association studies (TWAS) leverage expression quantitative trait loci (eQTL) and genome-wide association study (GWAS) data to identify novel susceptibility genes whose genetically predicted expression correlates with disease risk. However, no study has systematically integrated TWAS with scRNA-seq to unravel the pathogenesis of PAH at single-cell resolution. Using TWAS analysis, we identified a set of candidate genes genetically associated with PAH. We then evaluated the differential activity of these genes across PAH cell types at single-cell resolution using AUCell, Ucell, ssGSEA, and AddModuleScore algorithms. A subset of endothelial cells exhibiting elevated TWAS activity was identified via quartile-based stratification and designated as the high TWAS activity state (HTS) group. Multi-dimensional analyses, including observed-to-expected ratio (RO/E), CellChat, CytoTRACE, and scMetabolism, were employed to characterize the functional and communicative properties of HTS cells. Machine learning algorithms were integrated to identify signature genes of the HTS subpopulation, and a benchmarked random forest model was trained to predict HTS status. We performed immunohistochemistry and qRT-PCR validation of the signature genes (KLF2, RASIP1 and DEPP1) in PAH and control lung tissues to support their expression patterns. We demonstrated that HTS endothelial cells are strongly associated with PAH pathogenesis, exhibiting significant tissue tropism, enhanced roles in intercellular communication, and a progenitor-like function in endothelial differentiation. Machine learning-based feature selection revealed three robust signature genes: KLF2, RASIP1, and DEPP1. These genes demonstrated exceptional predictive power for identifying HTS cells, suggesting their potential as drivers of endothelial dysfunction in PAH. The random forest model, benchmarked against multiple algorithms, achieved high accuracy in predicting PAH progression using these genes. Immunohistochemical analysis of pulmonary artery and qRT-PCR result of lung tissues addressed the elevated expression of KLF2, RASIP1 and DEPP1 in arterial wall post-PAH. This study elucidates endothelial cell heterogeneity in PAH and establishes the central role of HTS cells in disease progression, cellular crosstalk, and developmental reprogramming. Our findings bridge the gap between GWAS and scRNA-seq methodologies and provide a transformative framework for understanding PAH mechanisms.

BACKGROUND:Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.METHODS:CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.RESULTS:Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.CONCLUSIONS:Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.

Abstract BACKGROUND Glioma stem cells (GSCs) have the capacity to transdifferentiate into endothelial-like cells, contributing to glioblastoma (GBM) progression and therapy resistance. However, the mechanisms driving GSC-derived endothelial cell (GDEC) formation remain poorly understood. METHODS We employed single-cell RNA sequencing (scRNA-seq) and immunofluorescence to identify GSC subpopulations. Comparative RNA-seq, scRNA-seq, and scATAC-seq were used to identify regulators of endothelial differentiation. To investigate upstream control of ROR1 expression, we conducted MeRIP-seq, RIP-seq, and RNA stability assays. Additionally, scRNA-seq, spatial transcriptomics, Co-IP, and co-culture assays were used to explore immune-mediated regulation. In vivo effects of ROR1 targeting were evaluated via AAV-BR1 delivery, transmission electron microscopy (TEM), and TUNEL assays. RESULTS scRNA-seq identified a CD133⁺/CD31⁺ GSC subpopulation in GBM, validated by immunofluorescence. Comparative transcriptomic analyses and public datasets (CCGA, TCGA) revealed ROR1 as a key mediator of GSC-to-GDEC differentiation. Multi-omics integration (scRNA-seq, scATAC-seq, spatial RNA-seq) and in situ validation confirmed ROR1’s central role. ROR1 overexpression promoted, while its knockdown suppressed, GDEC formation and tumor growth in mouse models. Furthermore, Hypoxia induced WTAP upregulation and immunosuppressive microenvironment plays a vital role in this process. Mechanistically, hypoxia upregulated WTAP expression in GSCs, which, via m6A modification, stabilized ROR1 through HuR binding.ROR1 then activated WNT signaling by binding WNT5A, as shown by WNT5A stimulation and co-IP assays. Single-cell and spatial transcriptomics revealed that WNT5A is primarily secreted by glioma-associated macrophages (GAMs), with WNT5A⁺ TAMs found in proximity to ROR1⁺ GDECs. Finally,in vivo, AAV-BR1-mediated ROR1 inhibition reduced tumor growth, normalized vasculature, and enhanced temozolomide (TMZ) delivery and efficacy, as confirmed by TEM, HPLC, and TUNEL assays. CONCLUSION Our findings uncover a hypoxia-induced WTAP-ROR1 axis that drives GSC transdifferentiation into GDECs via m6A-dependent stabilization of ROR1. WNT5A from GAMs activates ROR1-mediated WNT signaling, further promoting this process. Targeting ROR1 via AAV-BR1 normalizes vasculature and enhances TMZ sensitivity, offering a promising therapeutic strategy for GBM.

Enhanced regenerative potential of human dental pulp stem cells for the pulp-dentin complex through coculture with iPSC-derived endothelial cells: An in vitro study.

Odontogenic exosomes simulating the developmental microenvironment promote complete regeneration of pulp-dentin complex in vivo.

3D-printed nHA/PA66 porous scaffold: Regulating immune balance and vascularization synergistically promotes bone regeneration☆☆☆

An essential part of designing cardiovascular grafts is the fabrication of an artificial endothelial basement membrane (EBM) with the ability to support endothelial differentiation, especially under physiological dynamic forces. In this study, we introduce a novel artificial EBM constructed from a poly (xylitol sebacate) (PXS) polymer. First, the PXS prepolymer (pPXS) was blended with polyvinyl alcohol (PVA) at different ratios to achieve optimized production with a minimum amount of PVA to fabricate well-organized electrospun fiber networks of pPXS/PVA. Subsequently, pPXS/PVA was cross-linked at 120 °C under vacuum for two days to form a cPXS/PVA meshwork. Then, PVA and remaining pPXS were removed from the cPXS/PVA meshworks by serial rinsing in deionized water and ethanol to fabricate a defect-free fibrous sheet of cPXS. The fibrous cPXS sheets were characterized in terms of their structural, mechanical, and biological performance. The results confirmed that the cPXS sheets exhibited appropriate mechanical strength, acceptable wettability, ideal porosity, degradation behavior, and superior biocompatibility. Moreover, cPXS, as an artificial EBM, is capable of supporting endothelial differentiation of mesenchymal stem cells under dynamic culture conditions in a parallel plate bioreactor. Therefore, it can be inferred that fibrous cPXS sheet can be an ideal candidate for EBM tissue engineering and development of functional cardiovascular grafts.

Tooth extraction often leads to significant alveolar bone resorption, posing a major clinical challenge that compromises subsequent prosthodontic rehabilitation. This impaired bone regenerative capacity is primarily attributed to excessive reactive oxygen species (ROS), insufficient angiogenesis, and inadequate osteoinductive stimulation within the socket, collectively delaying the healing process. To address this, we developed an injectable dual-network hydrogel system loaded with metal-organic framework (MOF) and osteogenic growth peptide (OGP) to promote the tooth extraction socket healing. The double-network hydrogel, composed of gelatin methacryloyl (GelMA) and oxidized dextran (ODex), forms a dynamic network through the Schiff base reaction between the amino groups of GelMA and the aldehyde groups of ODex, enabling injectability and adaptation to the irregular shape of the socket, while the second covalent network, formed by photo-cross-linking, enhances its mechanical properties. Besides, MOF decomposes to release magnesium ion (Mg2+) and gallic acid (GA) to eliminate excess ROS and promote endothelial cell differentiation, facilitating angiogenesis, while OGP is an efficient osteoinductive agent. In vitro studies demonstrate that the composite hydrogel system efficiently exerts antioxidant properties to eliminate ROS, promotes angiogenesis, and enhances osteogenesis. Moreover, in vivo trials show that the composite hydrogel facilitates angiogenesis and bone formation in the tooth extraction socket, ultimately promoting the bone repair process.

Differential requirement of Hand1 during differentiation of lateral plate mesoderm lineages in Xenopus laevis.

Angiogenesis is essential for successful tissue regeneration, particularly in clinical contexts such as ischemic injury, wound healing, and reconstructive therapies. However, the establishment of functional vasculature remains a major limitation in organoid-based systems. In this study, we developed vascularized organoid tissue modules (Angio-TMs) by incorporating human umbilical vein endothelial cells (HUVECs) into scaffold-free, self-organized constructs. Remarkably, the inclusion of HUVECs at 1% of the total cell population was sufficient to generate highly reproducible and structurally stable Angio-TMs, which exhibited clear endothelial differentiation and vascular functionality both in vitro and in vivo. Furthermore, inhibition of transforming growth factor (TGF)-β signaling in Angio-TMs led to a 2.5-fold increase in vessel length density, demonstrating a substantial enhancement in angiogenic potential. These findings highlight Angio-TMs as a robust and modular platform for engineering vascularized tissues and underscore their translational relevance in regenerative medicine and tissue transplantation.

Angiogenic and reparative potency of a human cardiac CD90- mesenchymal subpopulation in heart ischemic model.

Epigenetics is increasingly recognized as a crucial factor in angiogenesis. Ubiquitin-like with PHD and RING Finger Domains 1 (UHRF1) is an important epigenetic regulatory protein involved in regulating cellular life processes, developing many diseases. However, its potential role in regulating embryonic vascular development and postnatal angiogenesis is unclear. Our study found that endothelial cell-specific UHRF1 knockout mice showed obvious developmental disorders at the embryonic stage (E11.5-15.5), including impaired development of the individual embryo size and organs, sparse vascularity in the yolk sac, or even death. In the lower limb ischemia model, UHRF1 expression in ischemic muscle tissues of mice is proportionate to the regeneration of blood vessels. To confirm the specific inhibition of UHRF1, we transfected an adeno-associated virus serotype 9 which inserted a TIE-2 promoter and mediated the delivery of short hairpin RNA (AAV9-TIE-2-shUHRF1) into mouse vascular endothelial cells to knock down UHRF1 specifically. We observed that the knockdown of UHRF1 in endothelial cells results in poorer lower limb perfusion in mice. Mechanically, UHRF1 knockdown decreased the tube-forming capacity of ECFCs, whereas overexpression of UHRF1 by diabetic ECFCs where UHRF1 expression is typically downregulated significantly increased the tube-forming capacity of the cells. RNAseq and related bioinformatics analyses showed that differentially expressed genes (DEGs) were mainly involved in angiogenesis-related pathways. The results of qPCR and western blot showed that the protein and mRNA levels of angiogenesis-related factors (VEGF, PDGF, and ANGPT1), as well as vascular endothelial surface marker molecules (VEGFR2, CD31, and c-Kit), were down-regulated accordingly. Furthermore, ChIP experiments showed that UHRF1 was able to bind the promoters of VEGFR2 and CD31, affecting the levels of histone-methylated protein (H3K4me3 and H3K27me3) enriched in the promoter region. However, the expression of CD31 and VEGFR2 can be reversed separately after the transformation of different histone-methylated protein levels (H3K4me3 and H3K27me3). Taken together, UHRF1 may regulate angiogenic gene expression and vascular endothelial cell differentiation through epigenetic mechanisms and is essential for angiogenesis.

Background: The emergence of functional endothelial and hematopoietic lineages from embryonic stem cells (ESCs) is fundamental to vascular and blood development. Vascular endothelial growth factor A (VEGF-A) is considered a pivotal regulator of angiogenesis, influencing endothelial progenitor cell proliferation, migration, and survival. Traditional two-dimensional (2D) culture systems have improved our understanding of early lineage commitment, but they do not fully capture the three-dimensional (3D) microenvironment that more closely mimics in vivo conditions. Recent studies have emphasized the importance of 3D culture platforms in promoting more physiologically relevant differentiation and cell–cell interactions, yet systematic comparisons of 2D versus 3D approaches in VEGF-mediated endothelial and hematopoietic differentiation remain limited. Methods: Mouse ESCs underwent a standardized mesoderm induction protocol beginning at day 0. After four days, Flk1 + multipotent progenitors were purified by fluorescence-activated cell sorting (FACS) and then cultured either in 2D adherent plates or within a 3D hydrogel matrix. Results: Flow cytometric analyses revealed that VEGF-A supplementation increased the percentage of CD144 + endothelial cells in both 2D and 3D cultures. However, the effect was markedly more pronounced in the 3D system, with a dose-dependent elevation in endothelial markers at VEGF-A concentrations ranging from 1 to 100 ng/mL. Concomitantly, hematopoietic lineage cells (CD41 + ) declined in the presence of VEGF-A, more substantially in 3D cultures, suggesting a shift in lineage commitment favoring an endothelial phenotype. Immunofluorescence staining confirmed robust CD144 expression and outlined the formation of tubular-like networks in 3D cultures receiving VEGF-A, whereas 2D monolayers displayed flattened endothelial cell morphologies with less pronounced cell–cell connections. Conclusion: Our findings indicate that 3D culture conditions enhance the pro-endothelial effects of VEGF-A on mouse ESC differentiation relative to conventional 2D systems. The 3D environment appears to mimic aspects of embryonic tissue architecture, allowing for improved cellular interactions and factor gradients that promote endothelial lineage specification. These results advance the understanding of how microenvironmental factors modulate vascular commitment and may provide a foundation for tissue engineering strategies that rely on robust vascularization.

Effects of prenatal azithromycin exposure at different stages, doses, and treatment durations on placental development and function in mice.

Mechanical forces stimulate human periodontal ligament stem cells (HPDLSCs) to release extracellular adenosine triphosphate (eATP). The eATP impacts various functions of HPDLSCs, i.e., immunosuppression and inflammation. eATP has been reported to promote the angiogenesis of pulmonary vascular endothelial cells. Shear force, one of the mechanical forces involved in orthodontic tooth movement, influences osteogenic differentiation and ECM remodeling of HPDLSCs. However, the relationship between shear force and the impact of eATP on endothelial differentiation and angiogenic characteristics of HPDLSCs remains unclear. This study aimed to determine the response of HPDLSCs on endothelial differentiation and angiogenic properties after shear stress loading and eATP treatment as well as explored the mechanism of eATP-involved in angiogenic responses of HPDLSCs. Shear stress application at 5 dyn/cm2 for 24h stimulated the release of ATP by HPDLSCs. Both shear stress and 200 µM eATP promoted the expression of endothelial differentiation and angiogenic markers (ANG1, CD31, EPCR, e-selectin, FLK1, TIE2, VEGF). Blockade of specific P2Y1 receptor and intracellular calcium signaling attenuated eATP-induced expression of endothelial differentiation and angiogenic expression. Both shear stress and eATP have stimulatory effects on endothelial differentiation and angiogenesis in HPDLSCs through P2Y1-intracellular calcium signaling.

Induced pluripotent stem cells (iPSCs) represent a promising source for regenerative therapies, yet allogeneic transplantation is limited by immune rejection. While strategies for generating hypoimmune iPSCs have been proposed, their efficacy after differentiation into lineage-specific cell types remains underexplored. A human iPSC line (36A) from peripheral blood mononuclear cells using a Sendai virus-based reprogramming protocol. Hypoimmune properties were conferred via CRISPR-Cpf1-mediated B2M knockout, combined with lentiviral overexpression of HLA-E and CD47. Immune evasion was validated using NK cell cytotoxicity assays. Endothelial differentiation was induced using a defined, stepwise protocol, and in vivo functionality was evaluated in humanized NSG mice. The hypoimmune iPSCs retained pluripotency, exhibited stable karyotype, and demonstrated > 99% expression of HLA-E/CD47. NK cell-mediated lysis was significantly reduced in edited cells, although IFN-γ levels remained elevated. Upon differentiation, the hypoimmune iPSCs yielded > 98% CD31+CD144+ endothelial cells, which showed enhanced survival in vivo compared to wild-type controls. Multiplex gene editing successfully conferred durable immune evasion in both undifferentiated and endothelial-differentiated iPSCs. These findings support the clinical potential of hypoimmune iPSC-derived cell therapies for allogeneic transplantation without immunosuppression.

The extracellular matrix (ECM) plays a vital role in stem cell differentiation to endothelial cells in vivo and is also important for the specification of endothelial cells in vitro. Individual ECM components have previously been shown to support endothelial differentiation; here, we use a Design of Experiments approach to optimize ECM composition to more effectively drive endothelial differentiation. We found that a combination of Collagen I, Collagen IV, and Laminin 411 could induce endothelial differentiation well beyond that found with Matrigel, the most commonly used differentiation substrate for endothelial cells. We also show that the addition of vascular endothelial growth factor (VEGF) during differentiation improves outcomes and that transforming growth factor beta (TGFβ) inhibits specification. The optimized ECM formulation (EO) was subsequently used to create bioprinted constructs, demonstrating its ability to spatially define endothelial differentiation in 3D environments. Our results build our mechanistic knowledge of the signaling axes that regulate differentiation in response to ECM stimulation with practical implications for the vascularization of engineered tissues.

MicroRNAs (miRNAs) play an important role in endothelial cell growth and differentiation. Tumor angiogenesis-specific miRNAs (angiomiRs) are a subset of miRNAs that are dyregulated in tumor endothelial cells. Because of the importance of angiogenesis in cancer progression, regulation of angiomiRs may have significant therapeutic implications. However, discovery of angiomiRs has often been limited by biased model systems that may not be valid. Here, we evaluated whether the variable expression levels of angiomiRs in endothelial cells were impacted by the isolation methods used to profile them. Using an autochthonous, genetically engineered mouse model of lung adenocarcinoma, we used Nanostring to profile miRNA expression levels of normal lung endothelial cells (NECs) to tumor endothelial cells (TECs) using two endothelial cell (EC) isolation methods: 1) staining and sorting ECs directly from tumors (" in vivo "), and 2) magnetic bead isolation and sub-cloning ECs (" in vitro "). We then compared candidate angiomiRs with the profiles from two orthotopic, immunocompetent lung cancer models. When TECs were directly enriched from tumors (" in vivo " method), three candidate angiomiRs (miR-30b, miR-1981, and miR-707) were significantly lower in TECs than NECs. In contrast, when ECs were isolated and cultured (" in vitro " method), three different candidate angiomiRs (miR-200a, miR-124 and miR-186) were significantly lower in TECs than NECs. Using two independent model systems for validation, we found miR-30b to be significantly reduced in TECs using freshly sorted ECs. Conversely, the in vitro discovered angiomiR candidates did not validate in these model systems, suggesting that TECs grown in vitro may not maintain relevant angiomiR profiles or serve as an adequate method for molecular profiling. Our findings demonstrate that angiomiR expression patterns are impacted by isolation methods. Instead of relying on ECs cultured in vitro , we suggest careful validation studies of cells freshly collected from tumors before determining whether a miRNA is a bona fide angiomiR.