OBJECTIVE: The objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs).
METHODS: We differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing.
RESULTS: GLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs.
CONCLUSIONS: CRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

Bisphosphonates are potent bone resorption inhibitors, among which alendronate sodium (ALN) is commonly prescribed for most osteoporosis patients, but long-term application of ALN can cause bisphosphonate-related osteonecrosis of jaw (BRONJ), the pathogenesis of which remains unclear. Previous studies have suggested that bisphosphonates cause jaw ischemia by affecting the biological behavior of vascular endothelial cells, leading to BRONJ. However, the impacts of ALN on vascular endothelial cells and its mechanism remain unclear. The purpose of this work is to assess the influence of ALN on human umbilical vein endothelial cells (HUVECs) and clarify the molecular pathways involved. We found that high concentration of ALN induced G1 phase arrest in HUVECs, demonstrated by downregulation of Cyclin D1 and Cyclin D3. Moreover, high concentration of ALN treatment showed pro-apoptotic effect on HUVECs, demonstrated by increased levels of the cleaved caspase-3, the cleaved PARP and Bax, along with decreased levels of anti-apoptotic protein Bcl-2. Further experiments showed that ERK1/2 phosphorylation was decreased. Additionally, ALN provoked the build-up of reactive oxygen species (ROS) in HUVECs, leading to ERK1/2 pathway suppression. N-acetyl-L-cysteine (NAC), a ROS scavenger, efficiently promoted the ERK1/2 phosphorylation and mitigated the G1 phase arrest and apoptosis triggered by ALN in HUVECs. PD0325901, an inhibitor of ERK1/2 that diminishes the ERK1/2 phosphorylation enhanced the ALN-induced G1 phase arrest and apoptosis in HUVECs. These findings show that ALN induces G1 phase arrest and apoptosis through ROS-mediated ERK1/2 pathway inhibition in HUVECs, providing novel insights into the pathogenic process, prevention and treatment of BRONJ in individuals receiving extended use of ALN.

Angiogenesis is crucial for successful bone defect repair. Co-transplanting Bone Marrow Stromal Cells (BMSCs) and Endothelial Cells (ECs) has shown promise for vascular augmentation, but it face challenges in hostile tissue microenvironments, including poor cell survival and limited efficacy. In this study, the mitochondria of human BMSCs are isolated and transplanted to BMSCs from the same batch and passage number (BMSCsmito). The transplanted mitochondria significantly boosted the ability of BMSCsmito-ECs to promote angiogenesis, as assessed by in vitro tube formation and spheroid sprouting assays, as well as in vivo transplantation experiments in balb/c mouse and SD rat models. The Dll4-Notch1 signaling pathway is found to play a key role in BMSCsmito-induced endothelial tube formation. Co-transplanting BMSCsmito with ECs in a rat cranial bone defect significantly improves functional vascular network formation, and improve bone repair outcomes. These findings thus highlight that mitochondrial transplantation, by acting through the DLL4-Notch1 signaling pathway, represents a promising therapeutic strategy for enhancing angiogenesis and improving bone repair. Hence, mitochondrial transplantation to BMSCS as a therapeutic approach for promoting angiogenesis offers valuable insights and holds much promise for innovative regenerative medicine therapies.

Oligodendrocyte precursor cells (OPCs) migrate extensively using blood vessels as physical scaffolds in the developing central nervous system. Although the association of OPCs with the vasculature is critical for migration, the regulatory mechanisms important for OPCs proliferative and oligodendrocyte development are unknown. Here, a correlation is demonstrated between the developing vasculature and OPCs response during brain development. Deletion of endothelial stimulator of interferon genes (STING) disrupts angiogenesis by inhibiting farnesyl-diphosphate farnesyltransferase 1 (FDFT1) and thereby reducing cholesterol synthesis. Furthermore, the perturbation of metabolic homeostasis in endothelial cells increases interleukin 17D production which mediates the signal transduction from endothelial cells to OPCs, which inhibits oligodendrocyte development and myelination and causes behavioral abnormalities in adult mice. Overall, these findings indicate how the endothelial STING maintains metabolic homeostasis and contributes to oligodendrocyte precursor cells response in the developing neocortex.

BACKGROUND: In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion.
METHODS: Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses.
RESULTS: Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group.
CONCLUSIONS: The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.

Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31hi Emcnhi ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31hi Emcnhi ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31hi Emcnhi ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31hi Emcnhi ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31hi Emcnhi ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.

Abnormal angiogenesis and increased vascular permeability of subchondral bone are key mechanisms related to osteoarthritis (OA). However, the precise mechanisms responsible for heightened vascular permeability in OA remain unclear. The present study used proteomics to identify protein expression in damaged subchondral bone compared with normal subchondral bone. The results suggest that Ras homolog family member A (RhoA) may be associated with the vascular permeability of subchondral bone and ferroptosis in OA. The results of analysis of clinical samples indicated a significant increase in expression of RhoA in the subchondral bone of OA. This were consistent with the proteomics findings. We found through western blotting, RT‑PCR, and immunofluorescence that RhoA significantly increased the permeability of endothelial cells (ECs) by inhibiting inter‑EC adhesion proteins (zona occludens‑1, connexin 43 and Vascular endothelial‑Cadherin) and actin filaments. Furthermore, RhoA induced ferroptosis core proteins (glutathione peroxidase 4,  solute carrier family 7 member 11 and acyl‑CoA synthase long‑chain family member 4, ACSL4) by influencing lipid peroxidation and mitochondrial function, leading to ferroptosis of ECs. This suggested an association between RhoA, ferroptosis and vascular permeability. Ferroptosis significantly increased permeability of ECs by inhibiting inter‑EC adhesion proteins. RhoA increased vascular permeability by inducing ferroptosis of ECs. In vivo, inhibition of RhoA and ferroptosis significantly mitigated progression of OA by alleviating cartilage degeneration and subchondral bone remodeling in mice with destabilization of the medial meniscus. In conclusion, the present findings indicated that RhoA enhanced vascular permeability in OA by inducing ferroptosis. This may serve as a novel strategy for the early prevention and treatment of OA.

Endothelial progenitor cells (EPCs) play a crucial role in maintaining vascular health and aiding in the repair of damaged blood vessels. However, the specific impact of EPCs-derived exosomes on vascular endothelial cell injury caused by lipopolysaccharide (LPS) remains inadequately understood. This study aims to explore the potential benefits of EPC-exosomes in mitigating LPS-induced vascular injury and to elucidate the underlying mechanism. Initially, EPCs were isolated from mouse peripheral blood, and their identity was confirmed through flow cytometry and immunocytochemistry. Subsequently, the exosomes derived from EPCs were identified using transmission electron microscopy (TEM) and western blot analysis. A sepsis model was induced by subjecting brain microvascular endothelial cells (BMECs) to LPS-induced injury. Both EPC and their exosomes demonstrated a significant increase in BMECs proliferation, reduced apoptosis, decreased levels of pro-inflammatory factors (TNF-α, IL-6, and caspase-3), and enhanced sprouting and angiogenesis of BMECs. Notable, the Exosomes demonstrated a more pronounced impact on these parameters. Furthermore, both EPCs and Exosomes exhibited significantly increased levels of miR-126a-5p, with the Exosomes showing a more substantial enhancement. These findings suggest that supplementing exosomal miR-126a-5p from EPCs can provide protective effects on BMECs, offering a potential therapeutic option for treating sepsis-induced microvascular endothelial cell injury.

The development of acute respiratory distress syndrome (ARDS) in sepsis is associated with substantial morbidity and mortality. However, the molecular pathogenesis underlying sepsis-induced ARDS remains elusive. Neutrophil heterogeneity and dysfunction contribute to uncontrolled inflammation in patients with ARDS. A specific subset of neutrophils undergoing reverse transendothelial migration (rTEM), which is characterized by an activated phenotype, is implicated in the systemic dissemination of inflammation. Using single-cell RNA sequencing (scRNA-seq), it identified functionally activated neutrophils exhibiting the rTEM phenotype in the lung of a sepsis mouse model using cecal ligation and puncture. The prevalence of neutrophils with the rTEM phenotype is elevated in the blood of patients with sepsis-associated ARDS and is positively correlated with disease severity. Mechanically, scRNA-seq and proteomic analys revealed that inflamed endothelial cell (EC) released extracellular vesicles (EVs) enriched in karyopherin subunit beta-1 (KPNB1), promoting abluminal-to-luminal neutrophil rTEM. Additionally, EC-derived EVs are elevated and positively correlated with the proportion of rTEM neutrophils in clinical sepsis. Collectively, EC-derived EV is identified as a critical regulator of neutrophil rTEM, providing insights into the contribution of rTEM neutrophils to sepsis-associated lung injury.

UNASSIGNED: Exertional heatstroke (EHS) mainly occurs in healthy young people with rapid onset and high mortality. EHS immune disorders can cause systemic inflammatory responses and multiple organ failure; however, the underlying mechanisms remain unclear. As high mobility group box 1 (HMGB1) is a prototypical alarmin that activates inflammatory and immune responses, this study aimed to investigate the effect and mechanism of HMGB1 in the pathogenesis of EHS.
UNASSIGNED: Peripheral blood mononuclear cell (PBMC) transcriptome sequencing of healthy volunteers, classical heatstroke patients, and EHS patients was performed. A mouse model of EHS was established and murine tissue damage was evaluated by H&E staining. HMGB1 localization and release were visualized using immunofluorescence staining. Human umbilical vein endothelial cells (HUVECs) and THP-1 cells were co-cultured to study the effects of HMGB1 on macrophages. A neutralizing anti-HMGB1 antibody was used to evaluate the efficacy of EHS treatment in mice.
UNASSIGNED: Plasma and serum HMGB1 levels were significantly increased in EHS patients or mice. EHS-induced endothelial cell pyroptosis promoted HMGB1 release in mice. HMGB1 derived from endothelial cell pyroptosis enhanced macrophage pyroptosis, resulting in immune disorders under EHS conditions. Administration of anti-HMGB1 markedly alleviated tissue injury and systemic inflammatory responses after EHS.
UNASSIGNED: The release of HMGB1 from pyroptotic endothelial cells after EHS promotes pyroptosis of macrophages and systemic inflammatory response, and HMGB1-neutralizing antibody therapy has good application prospects for EHS.