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Although it is an effective treatment for acute myeloid leukemia (AML), chemotherapy leads to myelosuppression and poor hematopoietic reconstruction. Hematopoiesis is regulated by bone marrow (BM) endothelial cells (ECs), and BM ECs are dysfunctional in acute leukemia patients with poor hematopoietic reconstitution after allogenic hematopoietic stem cell transplantation. Thus, it is crucial to explore the underlying mechanism of EC impairment and establish strategies for targeted therapy. TGF-β signaling was found to be upregulated in ECs from AML patients in complete remission (CR ECs) and led to CR EC damage. Administration of a TGF-β inhibitor rescued the dysfunction of ECs caused by TGF-β1 expression in vitro, especially their hematopoiesis-supporting ability. Moreover, inhibition of TGF-β expression repaired the BM EC damage triggered by chemotherapy in both AML patients in vitro and in an AML-CR murine model, and restored normal hematopoiesis without promoting AML progression. Mechanistically, our data reveal alterations in the transcriptomic pattern of damaged BM ECs, accompanied by the overexpression of downstream molecules TGF-βR1, pSmad2/3, and functional genes related to adhesion, angiogenesis suppression and pro-apoptosis. Collectively, our findings reveal for the first time that the activation of TGF-β signaling leads to BM EC dysfunction and poor hematopoietic reconstitution. Targeting TGF-β represents a potential therapeutic strategy to promote multilineage hematopoiesis, thereby benefiting more cancer patients who suffer from myelosuppression after chemotherapy., Competing Interests: Declaration of competing interest The authors wish to confirm that there are no known conflicts of interest associated with this publication. All co-authors have seen and approved this version of the manuscript and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Engineered neural tissue (EngNT) is a stabilised aligned cellular hydrogel that offers a potential alternative to the nerve autograft for the treatment of severe peripheral nerve injury. This work aimed to automate the production of EngNT, to improve the feasibility of scalable manufacture for clinical translation. Endothelial cells were used as the cellular component of the EngNT, with the formation of endothelial cell tube-like structures mimicking the polarised vascular structures formed early on in the natural regenerative process. Gel aspiration-ejection for the production of EngNT was automated by integrating a syringe pump with a robotic positioning system, using software coded in Python to control both devices. Having established the production method and tested mechanical properties, the EngNT containing human umbilical vein endothelial cells (EngNT-HUVEC) was characterised in terms of viability and alignment, compatibility with neurite outgrowth from rat dorsal root ganglion neurons and formation of endothelial cell networks in vitro . EngNT-HUVEC manufactured using the automated system contained viable and aligned endothelial cells, which developed into a network of multinucleated endothelial cell tube-like structures inside the constructs and an outer layer of endothelialisation. The EngNT-HUVEC constructs were made in various sizes within minutes. Constructs provided support and guidance to regenerating neurites in vitro . This work automated the formation of EngNT, facilitating high throughput manufacture at scale. The formation of endothelial cell tube-like structures within stabilised hydrogels provides an engineered tissue with potential for use in nerve repair., (Creative Commons Attribution license.)

Background: Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway., Methods: Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS -/- ) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF 165 a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care., Results: On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS -/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF 165 a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group)., Conclusions: In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder., Competing Interests: None.

This study examines KLF4's role in endothelial cells (ECs), emphasizing its effects on nitric oxide (NO) production, microvascular formation, and oxidative stress regulation following ischemic stroke. Through high-throughput sequencing, we identified eight cell subpopulations in carotid artery tissues post-stroke, with KLF4 notably elevated in ECs. KLF4 overexpression in ECs promoted NO synthesis, enhanced endothelial tube formation, mitigated oxidative stress, and improved smooth muscle cells (SMCs) function, collectively boosting blood flow in ischemic regions. These findings highlight KLF4 as pivotal in vascular regeneration and oxidative stress reduction, positioning it as a promising target for cardiovascular and cerebrovascular therapies., Competing Interests: Declaration of Competing Interest The author declares no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Ethnopharmacological Relevance: Ginseng (Panax ginseng C. A. Mey) is a common traditional Chinese medicine used for anti-inflammation, anti-apoptosis, anti-oxidative stress, and neuroprotection. Ginsenosides Rg1, the main active components isolated from ginseng, may be a feasible therapy for spinal cord injury (SCI)., Aims of the Study: SCI causes endothelial cell death and blood vessel rupture, ultimately resulting in long-term neurological impairment. As a result, encouraging spinal angiogenesis may be a feasible therapy for SCI. This investigation aimed to validate the capacity of ginsenoside Rg1 in stimulating angiogenesis within the spinal cord., Materials and Methods: Rats with SCI were injected intraperitoneally with ginsenoside Rg1. The effectiveness of ginsenoside Rg1 was assessed using the motor function score and the motor-evoked potential (MEP). Immunofluorescence techniques were applied to identify the spinal cord's angiogenesis. Angiogenic factors were examined through Western Blot (WB) and Immunohistochemistry. Oxygen-glucose deprivation (OGD) was employed to establish the hypoxia-ischemia model in vitro, and astrocytes (As) were given ginsenoside Rg1 and co-cultured with spinal cord microvascular endothelial cells (SCMECs). Immunofluorescence, wound healing test, and tube formation assay were used to identify the co-cultured SCMECs' activity. Finally, network pharmacology analysis and siRNA transfection were applied to verify the mechanism of ginsenoside Rg1 promoting angiogenesis., Results: The rats with SCI treated with ginsenoside Rg1 indicated more significant functional recovery, more pronounced angiogenesis, and higher levels of angiogenic factor expression. In vitro, the co-culture system with ginsenoside Rg1 intervention improved SCMECs' capacity for proliferating, migrating, and forming tubes, possibly by promoting the expression of vascular endothelial growth factor (VEGF) in As via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway., Conclusion: Ginsenoside Rg1 can regulate As to promote angiogenesis, which may help to understand the mechanism of promoting SCI recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cardiomyopathy is particularly common in septic patients. Our previous studies have shown that activation of the alpha 1 adrenergic receptor (α 1 -AR) on cardiomyocytes inhibits sepsis-induced myocardial dysfunction. However, the role of cardiac endothelial α 1 -AR in septic cardiomyopathy has not been determined. Here, we identified α 1 -AR expression in mouse and human endothelial cells and showed that activation of α 1 -AR with phenylephrine (PE) improved cardiac function and survival by preventing cardiac endothelial injury in septic mice. Mechanistically, activating α 1 -AR with PE decreased the expression of ICAM-1, VCAM-1, iNOS, E-selectin, and p-p38MAPK, while promoting PKC and ERK1/2 phosphorylation in LPS-treated endothelial cells. These effects were abolished by a PKC inhibitor or α 1 -AR antagonist. PE also reduced p65 nuclear translocation, but this suppression is not blocked by PKC inhibition. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on p38MAPK phosphorylation. Our results demonstrate that cardiac endothelial α 1 -AR activation prevents sepsis-induced myocardial dysfunction in mice by inhibiting the endothelial injury via PKC-ERK/p38MAPK signaling pathway and a PKC-independent inhibition of p65 nuclear translocation. These findings offer a new perspective for septic patients with cardiac dysfunction by inhibiting cardiac endothelial cell injury through α 1 -AR activation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cyclosporin A (CSA) is a potent immunosuppressive agent in pharmacologic studies. However, there is evidence for side effects, specifically in regard to vascular dysfunction. Its mode of action inducing endothelial cell toxicity is partially unclear, and a connection with an adverse outcome pathway (AOP) is not established yet. Therefore, we designed this study to get deeper insights into the mechanistic toxicology of CSA on angiogenesis. Stem cells, especially induced pluripotent stem cells (iPSCs) with the ability of differentiation to all organs of the body, are considered a promising in vitro model to reduce animal experimentation. In this study, we differentiated iPSCs to endothelial cells (ECs) as one cell type that in other studies would allow to generate cells or organoids from single donors. Flow cytometry and immunostaining confirmed our scalable differentiation protocol. Then dose and time course experiments assessing CSA cytotoxicity on iPS derived endothelial cells were performed. Transcriptomic data suggested CDA dependent induction of reactive oxygen species (ROS) and mitochondrial dysfunction, which was confirmed by in vitro experiments. Additionally, CSA impaired angiogenesis via ROS induction. Finally, we combined this information into an AOP, was developed based on here observed and literature based evidence for CSA-mediated endothelial cell toxicity. This AOP will help to design in vitro test batteries, model events observed in human toxicity studies, as well for predictive toxicology., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zahra Mazidi reports was provided by Evercyte GmbH. Zahra Mazidi reports a relationship with Evercyte GmbH that includes: employment., (Copyright © 2024. Published by Elsevier Ltd.)

Antimicrobial peptides (AMPs), particularly host defense peptides (HDPs), have gained recognition for their role in host defense mechanisms, but they have also shown potential as a promising anticancer, antiviral, antiparasitic, antifungal and immunomodulatory agent. Research studies in recent years have shown HDPs play a crucial role in endothelial cell function and biology. The function of endothelial cells is impacted by HDPs' complex interplay between cytoprotective and cytotoxic actions as they are known to modulate barrier integrity, inflammatory response and angiogenesis. This biphasic response varies and depends on the peptide structure, its concentration, and the microenvironment. These effects are mediated through key signaling pathways, including MAPK, NF-κB, and PI3K/Akt, which controls responses such as cell proliferation, apoptosis, and migration. In the present review, we have discussed the significance of the intriguing relationship between HDPs and endothelial cell physiology which suggests it potential as a therapeutic agents for the treating wounds, cardiovascular diseases, and inflammation-related endothelial damage., Competing Interests: Declaration of Competing Interest Authors declare no conflict of Interest., (Copyright © 2024. Published by Elsevier Inc.)

C1q/TNF-related protein 13 (CTRP13) is a secreted adipokine that has been shown to play an important role in a variety of cardiovascular diseases. However, the effect of CTRP13 on ferroptosis of endothelial cells and its underlying mechanism remain unclear. In the present study, we analyzed the effects of CTRP13 on endothelial dysfunction in high-lipid-induced ApoE -/- mice and ox-LDL-induced mouse aortic endothelial cells (MAECs). In vivo experiment: Male ApoE -/- mice fed high fat were given C1ql3 gene overexpression adeno-associated virus. The atherosclerotic plaque size, lipid content, collagen fiber proportion and iron deposition level were measured. In vitro, CTRP13 combined with ox-LDL was used to pretreat MAECs to detect cell survival rate, lipid peroxidation, iron ion deposition and mitochondrial level. In this study, CTRP13 was found to inhibit ferroptosis of endothelial cells, demonstrated by up-regulated the expression of ferroptosis protective protein glutathione Peroxidase 4 (GPX4), and decreased the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) protein. Mechanistically, gtp cyclohydrolase 1(GCH1) silencing or tetrahydrobiopterin (BH4) inhibiting may counteract the protective effect of CTRP13 on ox-LDL-induced ferroptosis of endothelial cells, which is characterized by decreased cell activity, mitochondrial damage, increased iron ion deposition and lipid peroxidation, decreased GPX4 expression, and increased ACSL4 expression. This study demonstrated for the first time that CTRP13 can improve mitochondrial oxidative stress, inhibit ferroptosis of endothelial cells and improve endothelial cell dysfunction by activating the GCH1/BH4 signaling pathway, thereby inhibiting the progression of atherosclerosis., (Copyright © 2024. Published by Elsevier B.V.)

Objective: Observing the effects of Tetramethylpyrazine (TMP) on the expression of Collagen IV and Laminin in neurovascular basement membrane and the apoptosis of vascular endothelial cells, and to study the mechanism of TMP in the treatment of sciatic nerve injury., Results: Compared with the NS group, the TMP group had a significant increase in the sciatic nerve function index (P < 0.01).The miss times in TMP group was significantly lower than that in NS group (P < 0.01). The HE staining results of the TMP group showed irregular arrangement of some neuronal axons and Schwann cells, and more edema and rupture of cells. The proliferation of glial cells and inflammatory cells was significantly increased in TMP group. The results of immunohistochemistry showed that the expression of type IV collagen and laminin in the TMP group group was distributed around the blood vessels, vascular endothelial cells, basal membrane and glial cells after SNI. The expression of type IV collagen and laminin in TMP group increased significantly(P < 0.05). Immunofluorescence showed that compared with NS group, the apoptosis rate of TMP group was significantly decreased (P < 0.01). Flow cytometry results showed that compared with the NS group, the number of CECs in the TMP group was significantly decreased (P < 0.01)., Conclusions: TMP can effectively improve the sciatic nerve functional index (SFI) of Sprague Dawley (SD) rats, enhance the proliferation of sciatic nerve vascular endothelial cells, reduce apoptosis, promote the expression of Collagen IV and Laminin in sciatic nerve microvascular basal membrane components, thereby promoting angiogenesis and improving lower limb function in rats., Competing Interests: Declarations Ethics approval and consent to participate The study was approved by the Institutional Animal Care and Use Committee of Southwest Medical University. Consent for publication Not applicable. Availability of data and material. Competing interests The authors declare no competing interests. Conflict of interest The authors declare no conflict of interest., (© 2024. The Author(s).)

In clinical practice, the effect of a high vault on corneal endothelial cells after implantable collamer lens (ICL) implantation remains unclear. Many clinicians theoretically assume that a high postoperative vault leads to rapid endothelial damage, but no study has yet proven this hypothesis. We conducted a paired-eye study to compare changes in corneal endothelial cell density (ECD) between high and low postoperative vault groups. This retrospective study included 150 eyes of 75 patients with bilateral postoperative vault levels differing by more than 200 μm after ICL implantation. Patients were followed up for 7 years with ECD measurements, and changes in ECD were assessed between 6 months and 7 years post-surgery. Over the 7-year follow-up period, the percentage of ECD loss was 15.04% and 14.45% in the high- and low-vault groups, respectively. The bilateral paired-eye comparison revealed a significant reduction in ECD in the high-vault group at 3, 5, and 7 years postoperatively (P-value < 0.001). In this paired-eye comparison of long-term observations, a higher vault was associated with greater ECD loss. Our study confirms that a high vault level may be an important risk factor for ECD loss following ICL implantation., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate Approval for this study was obtained from the Institutional Review Board of the Korean National Institute for Bioethics Policy. Informed consent was waived due to the retrospective nature of the study. All research procedures were conducted in accordance with the principles of the Declaration of Helsinki and KNIBP guidelines., (© 2024. The Author(s).)

Backgrounds: The role of miR-191-5p in cerebral ischemia-reperfusion (I/R) injury has been established, with its expression in endothelial cells demonstrating anti-angiogenic effects. A potential circular RNA, circRNA&#95;0003307, has been identified through bioinformatics analysis as a candidate for interaction with miR-191-5p, yet its functional significance in brain I/R injury remains unexplored. We aimed to investigate whether circRNA&#95;0003307 regulates brain microvascular endothelial cell (BMEC) vascular tube formation, invasion, and migration by regulating the miR-191-5p cascade., Methods: Mouse BMECs (bEnd.3) were culturedand exposed to oxygen-glucose deprivation (OGD). The effects of circRNA&#95;0003307 on vessel-like tube formation and cellular migration were examined. In addition, we investigated the protective effects of circRNA&#95;0003307 on I/R injury in mice., Results: The results showed the level of circRNA&#95;0003307 was concentration-dependently increased in OGD-induced bEnd.3 cells. ODG-induction enhanced angiogenesis, migration, and invasion of bEnd.3 cells, which were further promoted by the transfection of pcDNA-0003307. Silencing circRNA&#95;0003307 expression showed the opposite results. The dual luciferase assay demonstrated miRNA-191-5p interacted with circRNA&#95;00033073' UTR, and miRNA-191-5p could bind with CDK6. Meanwhile, circRNA&#95;0003307 promoted the expression of CDK6 by sponging miRNA-191-5p. The overexpression of circRNA&#95;0003307 activated the angiogenesis, migration, and invasion of OGD-induced bEnd.3 cells, which were hindered by miRNA-191-5p mimic or siRNA-CDK6. Thus, circRNA&#95;0003307 promoted ODG-induced angiogenesis, migration, and invasion of bEnd.3 cells by targeting miR-191-5p/CDK6 axis. In vivo, circRNA&#95;0003307 had protective effects on brain I/R injury, including neuroprotection, anti-apoptosis and angiogenesis., Conclusion: CircRNA&#95;0003307 may be a promisingtherapeutictarget forthe treatment of cerebral I/R injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Background: Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe -deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear., Methods: Apoe - or Ldlr -deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFβ (transforming growth factor β) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFβ1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root., Results: Both ubiquitous and VSMC-specific progerin expression in Apoe -null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFβ1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype., Conclusions: Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFβ1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis., Competing Interests: The authors report no conflicts. The funders had no role in the design of the study, the collection, analysis, or interpretation of data, and the reporting of the study.

Studying vascular responses to microgravity (MG) poses significant challenges in space medicine due to the limitations of conventional cell culture and animal models. To address these challenges, we have developed an innovative biosensory μvessel-gravity device that integrates organ-on-a-chip technology, 3D printing, and a 3D clinostat. This device enables cell interaction monitoring and flow shear stress modeling, thereby allowing accurate blood vessel cell sensory to changed mechanical environment. Our study reveals that simulated MG induces senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) within mono-cultured μvessels. Interestingly, co-culturing ECs and VSMCs in the μvessel mitigates EC senescence, although VSMC senescence remains unaffected. Furthermore, the application of continuous flow shear stress delays EC senescence and enhances tight junction integrity under MG conditions, underscoring the importance of incorporating mechanical factors into the device. Knocking down the mechanosensor Piezo1 in VSMCs delays senescence in both VSMCs and ECs under MG, highlighting the critical role of mechanosensors in vascular responses to MG. The biosensory μvessel-gravity device presents an innovative in vitro model designed to sense vascular changes induced by gravitational forces, effectively replicating the pro-aging effects of MG on vascular tissues. This holds significant potential for advancing research in aging-related vascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Vascular calcification is a significant factor contributing to the rupture of vulnerable atherosclerotic plaques, ultimately leading to cardiovascular disease. However, no effective treatments are currently available to slow the progression of vascular calcification. Notoginsenoside R1 (R1) and protocatechuic aldehyde (PCAD), primary active components extracted from Panax notoginseng and Salvia miltiorrhiza Burge, have shown potential in mitigating endothelial injury and atherosclerosis. This study investigated the effects of R1-PCAD on nitric oxide (NO) production in endothelial cells (ECs) and its role in counteracting vascular calcification and inflammation. Additionally, it explored the mechanisms underlying these effects. To simulate atherosclerotic calcification, apolipoprotein E-deficient (ApoE -/- ) mice were fed a high-fat diet and given intraperitoneal injections of vitamin D3. Treatment with the R1-PCAD combination improved endothelial function, reduced inflammation in the aorta, and lowered calcium deposition. Mechanistically, R1-PCAD enhanced eNOS-Ser1177 phosphorylation by activating the AMPKα/Akt pathway, which stimulated NO production and eNOS activation in ECs. In an in vitro co-culture model involving vascular smooth muscle cells (VSMCs) and ECs, R1-PCAD similarly reduced inflammation and calcification in VSMCs triggered by β-glycerophosphate, with these effects partially dependent on NO levels and EC functionality. Further investigation revealed that R1-PCAD facilitated NO release from ECs, which subsequently inhibited TGFβR1 activation in VSMCs. This inhibition reduced Smad2/3 activation and nuclear translocation of YAP/TAZ, thereby diminishing inflammation and calcification in VSMCs. These findings suggest that R1-PCAD alleviates vascular inflammation and calcification primarily via the NO-TGFβR1-YAP/TAZ signaling pathway. This study presents a promising new approach for treating vascular calcification by targeting intercellular signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Studies using human lung organoids (hLO) have focused on differentiation of lung epithelial subtypes into distal alveolar unit. A major question has been whether introducing endothelial cells (EC) and resultant vascularization alter development of hLO. We describe herein a method for vessel infiltration of hLO in which we determined differences of these hLOs with standard avascular hLOs. hLO are generated by combining hiPSC-derived lung progenitor cells (LP) with EC at different LP:EC ratios. This results in vascularization of hLO and enables comparisons with hLO generated without EC. We observe red blood-filled vessels in hLOs generated post-implantation into the kidney capsule of NOD/SCID mice. Both human and mouse EC conjoin in the capsule to form chimeric vessels in hLOs. Vessel-infiltrating hLOs show robust generation of alveolar type II epithelial cells (ATII) and alveolar type I cells (ATI), although there was no difference in the observed 1:1 ATII/ATI cell ratio. Electron microscopy revealed better-developed surfactant production apparatus in ATII of vascularized hLOs compared to avascular hLOs. We observed prominent primitive airway sacs with alveolar epithelial cells lining lumen in vascularized vs. avascular hLOs. The vessel-infiltrating hLOs also mounted a robust inflammatory response characterized by mouse PMN influx after challenging host mice with lipopolysaccharide. Thus, interaction of EC with LP generated vascularized hLOs and drive ATII and ATI differentiation and hLOs also mount a robust inflammatory response upon LPS challenge of hLO-transplanted recipient mice. Our results show usefulness of generating hLOs in studying human lung development and mechanisms underlying inflammatory lung injury.

Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect experienced by patients with lung cancer treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of proinflammatory cytokines, and neutrophil infiltration in the lungs of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing that TPOm reduced endothelial cell-neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition and expression of MMP-9, TIMP-1, IL-6, TGF-β, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells toward a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.

Background: EPHB4 (ephrin receptor B4) and the RASA1 (p120 Ras GTPase-activating protein) are necessary for the development of lymphatic vessel (LV) valves. However, precisely how EPHB4 and RASA1 regulate LV valve development is unknown. In this study, we examine the mechanisms by which EPHB4 and RASA1 regulate the development of LV valves., Methods: We used LV-specific inducible EPHB4-deficient mice and EPHB4 knockin mice that express a form of EPHB4 that is unable to bind RASA1 yet retains protein tyrosine kinase activity (EPHB4 2YP) to study the role of EPHB4 and RASA1 in LV valve development in the embryo and LV valve maintenance in adults. We also used human dermal lymphatic endothelial cells in vitro to study the role of EPHB4 and RASA1 as regulators of LV valve specification induced by oscillatory shear stress, considered the trigger for LV valve specification in vivo., Results: LV valve specification, continued valve development postspecification, and LV valve maintenance were blocked upon induced loss of EPHB4 in LV. LV valve specification and maintenance were also impaired in EPHB4 2YP mice. Defects in LV valve development were reversed by inhibition of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. In human dermal lymphatic endothelial cells, loss of expression of EPHB4 or its ephrin b2 ligand, loss of expression of RASA1, and inhibition of physical interaction between EPHB4 and RASA1 resulted in dysregulated oscillatory shear stress-induced Ras-MAPK activation and impaired expression of LV specification markers that could be rescued by Ras-MAPK pathway inhibition. The same results were observed when human dermal lymphatic endothelial cells were stimulated with the Yoda1 agonist of the PIEZO1 oscillatory shear stress sensor. Although Yoda1 increased the number of LV valves when administered to wild-type embryos, it did not increase LV valve number when administered to EPHB4 2YP embryos., Conclusions: EPHB4 is necessary for LV valve specification, continued valve development postspecification, and valve maintenance. LV valve specification requires physical interaction between EPHB4 and RASA1 to limit activation of the Ras-MAPK pathway in lymphatic endothelial cells. Specifically, EPHB4-RASA1 physical interaction is necessary to dampen Ras-MAPK activation induced through the PIEZO1 oscillatory shear stress sensor. These findings reveal the mechanism by which EPHB4 and RASA1 regulate the development of LV valves., Competing Interests: None.

Antithrombin and tissue factor pathway inhibitor (TFPI) provide different anticoagulant mechanisms. Having established a potent anticoagulant role of cultured human umbilical vein endothelial cells in vessel-on-a-chip microfluidic models, we now investigated how these cells modulated thrombin generation under stasis through antithrombin and TFPI pathways. We observed that endothelial monolayers in 96 well-plates strongly delayed and suppressed the thrombin generation process induced by tissue factor, regardless of the presence of whole blood, platelet-rich plasma or platelet-free plasma. Intervention studies indicated that the blocking of heparin-like proteoglycans with polybrene or protamine sulphate, similarly as the absence of antithrombin in plasma, reverted the endothelial anticoagulant activity. Heparinase treatment of the cells also reduced the anticoagulant potential. Furthermore, the presence of andexanet-alpha (inactivated factor Xa) and an anti-TFPI antibody were able to revert the endothelial effects. Jointly, these data point to additive anticoagulant mechanisms of endothelial cells through surface-expressed heparin-like proteoglycans and TFPI, both contributing to thrombin inhibition., Competing Interests: Declaration of competing interest H.M., D.H., M.R. and B.d.L. are employees of the Synapse Research Institute Maastricht (member of the Stago Diagnostic group), P.G.d.G. and J.W.M.H. are advisors of the same institute. The other authors do not report a conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The heterogeneity of endothelial cells (ECs) across human tissues remains incompletely inventoried. We constructed an atlas of > 210,000 ECs derived from 38 regions across 24 human tissues. Our analysis reveals significant differences in transcriptome, phenotype, metabolism and transcriptional regulation among ECs from various tissues. Notably, arterial, venous, and lymphatic ECs shared more common markers in multiple tissues than capillary ECs, which exhibited higher heterogeneity. This diversity in capillary ECs suggests their greater potential as targets for drug development. ECs from different tissues and vascular beds were found to be associated with specific diseases. Importantly, tissue specificity of EC senescence is more determined by somatic site than by tissue type (e.g. subcutaneus adipose tissue and visceral adipose tissue). Additionally, sex-specific differences in brain EC senescence were observed. Our EC atlas offers valuble resoursce for identifying EC subclusters in single-cell datasets from body tissues or organoids, facilitating the screen of tissue-specific targeted therapies, and serving as a powerful tool for future discoveries., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single-cell RNA sequencing (scRNA-seq) analysis of human islets exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory-macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared with separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory-macrophage-mediated β cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune-cell-mediated host damage and uncovered the mechanism of β cell damage during viral exposure., Competing Interests: Declaration of interests R.E.S. is on the scientific advisory board of Miromatrix Inc. and Lime Therapeutics and is a consultant and speaker for Alnylam Inc. S.C. and T.E are the co-founders of OncoBeat, LLC. S.C. is a consultant of Vesalius Therapeutics and co-founder of iOrganBio., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Highly complex tumor microenvironment makes hepatocellular carcinoma (HCC) as one of the most malignant tumors worldwide. The role of cellular senescence in HCC has been gradually recognized. The present study aimed to comprehensively elucidate the senescence-related features of HCC in single-cell and spatial dimension., Methods: Single-cell RNA sequencing (scRNA-Seq) data was used to clarify the heterogeneity of senescence-related genes (SRGs) among multiple cell types within HCC. Spatial transcriptome RNA sequencing (stRNA-Seq) data was used for depicting SRGs features in spatial dimension. A prognostic model based on SRGs was constructed by using of bulk sequencing (bulk-Seq) data of HCC. The cell-cell interaction of senescent endothelial cells (ECs) in tumor microenvironment was analyzed. Then, the role of senescent ECs was verified through in vitro and in vivo experiments., Results: The level of senescence demonstrated substantial heterogeneity among different cell types within tumor microenvironment of HCC, where ECs exhibited the most prominent senescent phenotype. Senescent ECs activated specific regulatory pathways through communicating with other cell types, with a potential impact on tumor progression. Spatial analysis revealed senescent ECs mainly located in the core region of HCC. The interaction of senescent ECs and immune cells implicated their role in tumor progression and immunotherapeutic response. In addition, CDKN2A was identified as an independent risk factor for HCC prognosis by constructing a prognostic model. Patients with high risk displayed an even worse outcome. The experimental verification indicated senescence of ECs determined by CDKN2A exhibited a secretory phenotype. Furthermore, senescent ECs with CDKN2A overexpression promote the proliferation and migration of HCC., Conclusion: The present study recognizes the critical effect of senescent ECs defined by CDKN2A in the promotion of tumor progression, which sheds new light on the investigation of ECs senescence in HCC., (© 2024. The Author(s).)

To maintain homeostasis in the heart, endothelial cells and cardiomyocytes engage in dynamic cross-talk through paracrine signals that regulate both cardiac development and function. Here, we review the paracrine signals that endothelial cells release to regulate cardiomyocyte growth, hypertrophy and contractility, and the factors that cardiomyocytes release to influence angiogenesis and vascular tone. Dysregulated communication between these cell types can drive pathophysiology of disease, as seen in ischemia-reperfusion injury, diabetes, maladaptive hypertrophy, and chemotherapy-induced cardiotoxicity. Investingating the role of cross-talk is critical in developing an understanding of tissue homeostasis, regeneration, and disease pathogenesis, with the potential to identify novel targets for diagnostic and therapeutic purposes., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for in vivo gene delivery. We screened a library of >1,500 US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, as documented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. In vivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of VWF. The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients., Objective: This study aims to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex-vivo cell models., Methods: ECFCs were isolated from venous blood of VWD patients from the Willebrand in the Netherlands (WiN) cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord-blood derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy., Results: Two unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired proVWF processing into mature VWF with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum (ER); this was confirmed in various populations of base edited CB-ECFCs harboring the p.M771V variant., Conclusion: The severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modelling system, show that ER retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The functional and structural integrity of the endothelium is essential for vascular homeostasis. Loss of barrier function in quiescent and migratory capacity in proliferative endothelium causes exuberant vascular permeability, a cardinal feature of many inflammatory diseases including acute lung injury (ALI). However, the signals governing these fundamental endothelial cell (EC) functions are poorly understood. Here, we identify Mechanistic Target of Rapamycin (MTOR) as an important link in preserving the barrier integrity and migratory/angiogenic responses in EC and preventing lung vascular injury and mortality in mice. Knockdown of MTOR in EC altered cell morphology, impaired proliferation and migration, and increased endocytosis of cell surface VE-Cadherin leading to disrupted barrier function. MTOR-depleted EC also exhibited reduced VE-Cadherin and VEGFR2 levels mediated in part by autophagy. Similarly, lungs from mice with EC-specific MTOR deficiency displayed spontaneous vascular leakage marked by decreased VE-Cadherin and VEGFR2 levels, indicating that MTOR deficiency in EC is sufficient to disrupt lung vascular integrity and may be a key pathogenic mechanism of ALI. Indeed, MTOR as well as VEGFR2 and VE-Cadherin levels were markedly reduced in injured mouse lungs or EC. Importantly, EC-targeted gene transfer of MTOR cDNA, either prophylactically or therapeutically, mitigated inflammatory lung injury, and improved lung function and survival in mouse models of ALI. These findings reveal an essential role of MTOR in maintaining EC function, identify loss of endothelial MTOR as a key mechanism of lung vascular injury, and show the therapeutic potential of EC-targeted MTOR expression in combating ALI and mortality in mice., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Disturbed flow generates oscillatory shear stress (OSS), which in turn leads to endothelial inflammation and atherosclerosis. Piezo1, a biomechanical force sensor, plays a crucial role in the cardiovascular system. However, the specific role of Piezo1 in atherosclerosis remains to be fully elucidated., Methods and Results: We detected the expression of Piezo1 in atherosclerotic mice and endothelial cells from regions with disturbed blood flow. The pharmacological inhibitor Piezo1 inhibitor (GsMTx4) was used to evaluate the impact of Piezo1 on plaque progression and endothelial inflammation. We examined Piezo1's direct response to OSS in vitro and its effects on endothelial inflammation. Furthermore, mechanistic studies were conducted to explore the potential molecular cascade through which Piezo1 mediates endothelial inflammation in response to OSS. Our findings revealed the upregulation of Piezo1 in apoE-/- (apolipoprotein E) atherosclerotic mice, which is associated with disturbed flow. Treatment with GsMTx4 not only delayed plaque progression but also mitigated endothelial inflammation in both chronic and disturbed flow-induced atherosclerosis. Piezo1 was shown to facilitate calcium ions (Ca 2 + ) influx in response to OSS, thereby activating endothelial inflammation. This inflammatory response was attenuated in the absence of Piezo1. Additionally, we identified that under OSS, Piezo1 activates the Ca 2 + /CaM/CaMKII (calmodulin/calmodulin-dependent protein kinases Ⅱ) pathways, which subsequently stimulate downstream kinases FAK (focal adhesion kinase) and Src. This leads to the activation of the OSS-sensitive YAP (yes-associated protein), ultimately triggering endothelial inflammation., Conclusions: Our study highlights the key role of Piezo1 in atherosclerotic endothelial inflammation, proposing the Piezo1-Ca 2+ /CaM/CaMKII-FAK/Src-YAP axis as a previously unknown endothelial mechanotransduction pathway. Piezo1 is expected to become a potential therapeutic target for atherosclerosis and cardiovascular diseases.

Endothelial cells (ECs) not only form passive blood conduits but actively contribute to nutrient transport and organ homeostasis. The role of ECs in glucose homeostasis is, however, poorly understood. Here, we show that, in skeletal muscle, endothelial glucose transporter 1 (Glut1/Slc2a1) controls glucose uptake via vascular metabolic control of muscle-resident macrophages without affecting transendothelial glucose transport. Lowering endothelial Glut1 via genetic depletion (Glut1 ΔEC ) or upon a short-term high-fat diet increased angiocrine osteopontin (OPN/Spp1) secretion. This promoted resident muscle macrophage activation and proliferation, which impaired muscle insulin sensitivity. Consequently, co-deleting Spp1 from ECs prevented macrophage accumulation and improved insulin sensitivity in Glut1 ΔEC mice. Mechanistically, Glut1-dependent endothelial glucose metabolic rewiring increased OPN in a serine metabolism-dependent fashion. Our data illustrate how the glycolytic endothelium creates a microenvironment that controls resident muscle macrophage phenotype and function and directly links resident muscle macrophages to the maintenance of muscle glucose homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aim and Objective: Our recent report showed that soluble T-cadherin promotes pancreatic beta-cell proliferation. However, how and where the secretion of soluble T-cadherin is regulated remain unclear., Methods and Results: Soluble T-cadherin levels significantly increased in leptin receptor-deficient db/db mice with hypoinsulinaemia or in wild-type mice treated with insulin receptor blockade by S961. Similar results were observed in human subjects; Diabetic ketoacidosis patients at the time of hospitalization had increased plasma soluble T-cadherin levels, which decreased after insulin infusion therapy. Patients with recurrent ovarian cancer who were administered a phosphatidylinositol-3 kinase (PI3K)-alpha inhibitor (a new anticancer drug) had increased plasma soluble T-cadherin and plasma C-peptide levels. Endothelial cell-specific T-cadherin knockout mice, but not skeletal muscle- or cardiac muscle-specific T-cadherin knockout mice, showed a 26 % reduction in plasma soluble T-cadherin levels and a significant increase in blood glucose levels in streptozocin-induced diabetes. The secretion of soluble T-cadherin from human endothelial cells was approximately 20 % decreased by insulin and this decrease was canceled by blockade of insulin receptor/Akt signalling, not Erk signalling., Conclusion: We conclude that insulin regulates soluble T-cadherin levels and soluble T-cadherin secretion from endothelial cells is positively regulated by insulin/insulin receptor/Akt signalling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Chronic hypoxia causes detrimental structural alterations in the lung, which may cause pulmonary hypertension and are partially mediated by the endothelium. While its relevance for the development of hypoxia-associated lung diseases is well known, determinants controlling the initial adaptation of the lung endothelium to hypoxia remain largely unexplored., Methods and Results: We revealed that hypoxia activates the transcription factor nuclear factor of activated T-cells 5 (NFAT5) and studied its regulatory function in murine lung endothelial cells (MLECs). EC-specific knockout of Nfat5 (Nfat5(EC)-/-) in mice exposed to normobaric hypoxia (10% O2) for 21 days promoted vascular fibrosis and aggravated the increase in pulmonary right ventricular systolic pressure as well as right ventricular dysfunction as compared with control mice. Microarray- and single-cell RNA-sequencing-based analyses revealed an impaired growth factor-, energy-, and protein-metabolism-associated gene expression in Nfat5-deficient MLEC after exposure to hypoxia for 7 days. Specifically, loss of NFAT5 boosted the expression and release of platelet-derived growth factor B (Pdgfb)-a hypoxia-inducible factor 1 alpha (HIF1α)-regulated driver of vascular smooth muscle cell (VSMC) growth-in capillary MLEC of hypoxia-exposed Nfat5(EC)-/- mice, which was accompanied by intensified VSMC coverage of distal pulmonary arteries., Conclusion: Collectively, our study shows that early and transient subpopulation-specific responses of MLEC to hypoxia may determine the degree of organ dysfunction in later stages. In this context, NFAT5 acts as a protective transcription factor required to rapidly adjust the endothelial transcriptome to cope with hypoxia. Specifically, NFAT5 restricts HIF1α-mediated Pdgfb expression and consequently limits muscularization and resistance of the pulmonary vasculature., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Pulmonary hypertension, characterized by vascular remodeling, currently lacks curative therapeutic options. The dysfunction of pulmonary artery endothelial cells plays a pivotal role in the initiation and progression of pulmonary hypertension (PH). ErbB3 (human epidermal growth factor receptor 3), also recognized as HER3, is a member of the ErbB family of receptor tyrosine kinases., Methods: Microarray, immunofluorescence, and Western blotting analyses were conducted to investigate the pathological role of ErbB3. Blood samples were collected for biomarker examination from healthy donors or patients with hypoxic PH. The pathological functions of ErbB3 were further validated in rodents subjected to chronic hypoxia- and Sugen-induced PH, with or without adeno-associated virus-mediated ErbB3 overexpression, systemic deletion, or endothelial cell-specific ErbB3 knockdown. Primary human pulmonary artery endothelial cells and pulmonary artery smooth muscle cells were used to elucidate the underlying mechanisms., Results: ErbB3 exhibited significant upregulation in the serum, lungs, distal pulmonary arteries, and pulmonary artery endothelial cells isolated from patients with PH compared with those from healthy donors. ErbB3 overexpression stimulated hypoxia-induced endothelial cell proliferation, exacerbated pulmonary artery remodeling, elevated systolic pressure in the right ventricle, and promoted right ventricular hypertrophy in murine models of PH. Conversely, systemic deletion or endothelial cell-specific knockout of ErbB3 yielded opposite effects. Coimmunoprecipitation and proteomic analysis identified YB-1 (Y-box binding protein 1) as a downstream target of ErbB3. ErbB3 induced nuclear translocation of YB-1 and subsequently promoted hypoxia-inducible factor 1/2α transcription. A positive loop involving ErbB3-periostin-hypoxia-inducible factor 1/2α was identified to mediate the progressive development of this disease. MM-121, a human anti-ErbB3 monoclonal antibody, exhibited both preventive and therapeutic effects against hypoxia-induced PH., Conclusions: Our study reveals, for the first time, that ErbB3 serves as a novel biomarker and a promising target for the treatment of PH., Competing Interests: None.

Background: Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability., Methods: Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice ( Kindlin-2 iΔEC ) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm 2 ) or oscillatory shear (0.5±4 dynes/cm 2 ) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function., Results: We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2 iΔEC mice showed heightened vascular permeability. Kindlin-2 iΔEC were bred onto ApoE -/- mice to generate Kindlin-2 iΔEC ; ApoE - /- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation., Conclusions: Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis., Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

In this study, we propose a new method for bioprinting 3D Spheroids to study complex congenital heart disease known as discrete subaortic stenosis (DSS). The bioprinter allows us to manipulate the extrusion pressure to change the size of the spheroids, and the alginate porosity increases in size over time. The spheroids are composed of human umbilical vein endothelial cells (HUVECs), and we demonstrated that pressure and time during the bioprinting process can modulate the diameter of the spheroids. In addition, we used Pluronic acid to maintain the shape and position of the spheroids. Characterization of HUVECs in the spheroids confirmed their uniform distribution and we demonstrated cell viability as a function of time. Compared to traditional 2D cell cultures, the 3D spheroids model provides more relevant physiological environments, making it valuable for drug testing and therapeutic applications., (© 2024. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) during mouse embryogenesis. Understanding the signaling molecules required for HSC development is crucial for the in vitro derivation of HSCs. We previously induced HSCs from embryonic HECs, isolated at embryonic day 10.5 (E10.5), in serum-free culture conditions with stem cell factor, thrombopoietin, and an endothelial feeder layer. Here, we aimed to elucidate signal requirements for inducing HSCs from earlier-stage HECs. Single-cell RNA sequencing (RNA-seq) analysis detected bone morphogenetic protein (BMP) signaling activation in E9.5 HECs. Adding BMP4 to the culture conditions led to the induction of HSCs from E9.5 HECs. Furthermore, isolating BMP4 receptor-expressing HECs from E9.5 embryos enriched progenitors with HSC-forming ability. This study identified BMP4 as an essential factor promoting the differentiation of early HECs into HSCs, opening up new possibilities for the in vitro derivation of HSCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Suppression of Tumorigenicity 2 (ST2) is a member of the interleukin-1 receptor/ Toll-like receptor superfamily, and its specific ligand is Interleukin-33 (IL-33). IL-33/ ST2 signaling has been implicated in numerous inflammatory and allergic diseases, as well as in promoting malignant behavior of tumor cells and angiogenesis. However, the precise role of ST2 in gastric cancer angiogenesis remains incompletely elucidated. We observed a significant correlation between high expression of ST2 in gastric cancer tissues and poor prognosis, along with various clinicopathological features. In vitro experiments demonstrated that the IL-33/ ST2 axis activates the PI3K/AKT/NF-κB signaling pathway through TRAF6, thereby promoting VEGFA-mediated tumor angiogenesis; meanwhile sST2 acts as a decoy receptor to regulate the IL-33/ST2L axis. Consistent findings were also observed in subcutaneous xenograft tumor models in nude mice. Furthermore, we investigated the molecular mechanism by which IL-33 promotes ST2L expression in GC cells via upregulation of transcription factors YY1 and GATA2 through intracellular signaling pathways., (© 2024. The Author(s).)

Background: Enhanced angiogenesis following myocardial infarction (MI) is beneficial to preserve cardiac function. The present study aimed to investigate whether acetylated derivatives of cordycepin altered its original antitumor properties and exerted cardioprotective effects by promoting angiogenesis in vitro and in vivo ., Methods: Cordycepin and its derivatives with single (DA), double (DAA), and triple acetyl groups (DAAA) were assessed. The cell viability of leukemia U937 cells, malignant hepatoma Huh-7 cells, and human umbilical vascular endothelial cells (HUVECs) treated with cordycepin, DA, DAA, and DAAA were determined. The expression of β-catenin in U937 cells, as well as the expression of p65, p38 and other related signal regulators in HUVECs elicited by lipopolysaccharides (LPS) were also observed. Angiogenesis was determined by tube formation in HUVECs and Matrigel plug assay in mice. Cardiac function following administration of DAAA was evaluated in mice MI model simulated by coronary artery ligation., Results: The inhibitory effects of cordycepin and its acetylated derivatives on U937 cells, Huh-7 cells, HUVECs, and the expression of β-catenin in U937 cells were mitigated with increasing acetylation. Intriguingly, DAAA preserved the cell viability of HUVECs compared to other acetylated derivatives. Although DAAA had a significantly diminished antitumor effect compared to cordycepin, it promoted angiogenesis in mice and tube formation in HUVECs and attenuated LPS-induced phosphorylation of p65 and p38. Additionally, administration of DAAA improved cardiac function following coronary artery ligation in mice., Conclusion: DAAA could be considered a promising adjunctive therapy to prevent post-MI heart failure through promoting angiogenesis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Myocardial infarction (MI) is a critical global health issue and a leading cause of heart failure. Indeed, while neonatal mammals can regenerate cardiac tissue mainly through cardiomyocyte proliferation, this ability is lost shortly after birth, resulting in the adult heart's inability to regenerate after injury effectively. In adult mammals, the adverse cardiac remodelling, which compensates for the loss of cardiac cells, impairs cardiac function due to the non-contractile nature of fibrotic tissue. Moreover, the neovascularisation after MI is inadequate to restore blood flow to the infarcted myocardium. This review aims to synthesise the most recent insights into the molecular and cellular players involved in endogenous myocardial and vascular regeneration, facilitating the identification of mechanisms that could be targeted to trigger cardiac regeneration, reduce fibrosis, and improve functional recovery post-MI. Reprogramming adult cardiomyocytes to regain their proliferative potential, along with the modulation of target cells responsible for neovascularisation, represents promising therapeutic strategies. An updated overview of endogenous mechanisms that regulate both myocardial and coronary vasculature regeneration-including stem and progenitor cells, growth factors, cell cycle regulators, and key signalling pathways-could help identify new critical intervention points for therapeutic applications.

Sepsis-induced myocardial dysfunction presents significant challenges in clinical management and is associated with increased mortality. Anisodamine (654-1/-2) has potentials in alleviating cardiac and endothelial impairments associated with sepsis. Exosomes, small vesicles secreted by cells, carry various bioactive molecules, such as nucleic acids, proteins, and lipids. These vesicles can travel to target cells to influence their function and modulating biological processes. In the context of endothelial-cardiac crosstalk, exosomes derived from endothelial cells can transfer signals that either exacerbate or mitigate myocardial injury, playing a crucial role in the progression of cardiovascular diseases. However, the precise role of endothelial-cardiac crosstalk, particularly through exosomes, in mediating the cardioprotective effects of anisodamine remains unclear. This study evaluated the effects of anisodamine on myocardial and endothelial injuries induced by LPS. Mechanisms were analyzed through network pharmacology, molecular docking, Western blotting, and RT-qPCR. The interaction between endothelial and cardiomyocyte inflammatory responses to anisodamine was assessed using a co-culture assay. Furthermore, both in vivo and in vitro assays were conducted to evaluate the effects of anisodamine-/LPS- treated HUVECs exosomes on A16 cell and myocardial function in mice. Anisodamine effectively mitigated apoptosis, inflammation, mitochondrial and myocardial injury, glycocalyx degradation, and oxidative stress by regulating the PI3K-AKT, NLRP-3/Caspase-1/ASC, TNF-α/PKCα/eNOs/NO, and NF-κB/iNOs/NO pathways in A16 cells and HUVECs. Moreover, in vivo and in vitro assays confirmed the protective effects of anisodamine against myocardial injuries mediated by exosomes derived from LPS-treated HUVECs. In summary, anisodamine ameliorated inflammation-induced endothelial and cardiomyocyte dysfunction. The in vitro and in vivo assays demonstrated that anisodamine could alleviate myocardial dysfunction through exosome-mediated mechanisms, offering new therapeutic avenues for treating myocardial injury and highlighting the potential of targeted exosome therapy in clinical settings., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The hypersecretion of cytokines triggers life-threatening systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction syndrome (MODS) and mortality. Although both coagulopathy and necroptosis have been identified as important factors in the pathogenesis of SIRS, the specific cell types that undergo necroptosis and the interrelationships between coagulopathy and necroptosis remain unclear. In this study, we utilized visualization analysis via intravital microscopy to demonstrate that both anticoagulant heparin and nonanticoagulant heparin (NAH) pretreatment protect mice against TNF-α-induced mortality in SIRS. Moreover, the deletion of Mlkl or Ripk3 resulted in decreased coagulation and reduced mortality in TNF-α-induced SIRS. These findings suggest that necroptosis plays a key role upstream of coagulation in SIRS-related mortality. Furthermore, using a genetic lineage tracing mouse model (Tie2-Cre;Rosa26-tdT), we tracked endothelial cells (ECs) and verified that EC necroptosis is responsible for the vascular damage observed in TNF-α-treated mice. Importantly, Mlkl deletion in vascular ECs in mice had a similar protective effect against lethal SIRS by blocking EC necroptosis to protect the integrity of the endothelium. Collectively, our findings demonstrated that RIPK3-MLKL-dependent necroptosis disrupted vascular integrity, resulting in coagulopathy and multiorgan failure, eventually leading to mortality in SIRS patients. These results highlight the importance of targeting vascular EC necroptosis for the development of effective treatments for SIRS patients., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Tissue kallikrein (TK) has emerged as a potential neuroprotective agent in ischemic stroke (IS), yet the optimal timing and mechanisms of TK therapy remain unclear. Here, we established a causal link between lower baseline TK levels and an increased risk of stroke through a retrospective, multicenter cohort study involving 2115 initially non-stroke subjects monitored for 5 years. Sequentially, we observed a notable increase in bradykinin receptor 2 (B2R) levels during the ischemic phase of the IS model, while levels of TK and bradykinin receptor 1 (B1R) remained stable. Intriguingly, both B1R and B2R exhibited a significant elevation 24 h after reperfusion. Further investigations in preclinical models demonstrated that TK supplementation activates the PI3K/AKT signaling pathway via enhanced B2R expression during the ischemic phase, leading to nuclear translocation of Hif-1α. This activation enhances the expression of VEGF and eNOS, thereby fortifying the neurovascular unit. Moreover, it suppresses the activation of the kallikrein-kinin system induced by reperfusion injury, effectively reducing inflammation, ROS production, apoptosis, and endothelial barrier dysfunction. Thus, our findings highlight the significance of TK supplementation during the ischemic phase in attenuating reperfusion-induced injury in IS, providing a mechanistic rationale for determining the optimal timing for TK supplementation therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~ 90% of syndromic and non-syndromic CM specimens and is present in CD31 pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimens from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1 and claudin-5, and were surrounded by MRC1 pos /LYVE1 pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose of Review: Hypoplastic left heart syndrome (HLHS) is a critical congenital heart defect characterized by the underdevelopment of left-sided heart structures, leading to significant circulatory challenges, and necessitating multiple surgeries for survival. Despite advancements in surgical interventions, long-term outcomes often involve heart failure, highlighting the need for a deeper understanding of HLHS pathogenesis. Current in vivo and in vitro models aim to recapitulate HLHS anatomy and physiology, yet they face limitations in accuracy and complexity., Recent Findings: In vivo models, including those in chick, lamb, and mouse, provide insights into hemodynamic and genetic factors influencing HLHS. In vitro models using human induced pluripotent stem cells offer valuable platforms for studying genetic mutations and cellular mechanisms. This review evaluates these models' utility and limitations, and proposes future directions for developing more sophisticated models to enhance our understanding and treatment of HLHS., (© 2024. The Author(s).)

Background: Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 (calcium release-activated calcium modulator 1) is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 (Ras-proximate-1) isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation., Methods: The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches., Results: We explored Ca 2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 μmol/L BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), 10 μmol/L GSK-7975A, and 5 μmol/L Gd 3+ . Whole-cell patch clamp measurements revealed enhanced Ca 2+ release-activated Ca 2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1 [C-X-C motif chemokine ligand 1], CXCL11 [C-X-C motif chemokine 11], CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1A iΔEC ) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1A iΔEC mice., Conclusions: Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca 2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions., Competing Interests: None.

Background: Under normal circumstances, high-density lipoprotein (HDL) is considered to have cardiovascular protective effects, but the impact of oxidized HDL (ox-HDL) on vascular endothelial function remains poorly understood. Mitochondrial function is closely related to endothelial function, and hydrogen sulfide (H₂S) is a gas with endothelial protective properties. The novel hydrogen sulfide donor AP39 can target mitochondria to release H₂S, but the combined effects of ox-HDL and AP39 on vascular endothelium are not well studied., Methods: We established a cell model of ox-HDL-induced endothelial cell damage and mitochondrial dysfunction using human umbilical vein endothelial cells (HUVECs) and conducted AP39 pretreatment. The experiments confirmed the functional damage and mitochondrial dysfunction in HUVECs caused by ox-HDL. Additionally, to further explore the role of SIRT1 in AS, we analyzed SIRT1 expression in AS carotid artery tissue. This included the analysis of differentially expressed genes from AS-related datasets, presented through volcano plots and heatmaps, with enrichment analysis of downregulated genes in KEGG pathways and GO functions. Furthermore, we evaluated the differences in SIRT1 expression in coronary arteries with varying degrees of stenosis and in early and late-stage AS carotid artery tissues, and analyzed data from SIRT1 knockout mouse models., Results: The experimental results indicate that AP39 effectively alleviated ox-HDL-induced endothelial cell damage and mitochondrial dysfunction by upregulating SIRT1 expression. MTT and CCK-8 assays showed that ox-HDL treatment led to decreased cell viability and proliferation in HUVECs, reduced eNOS expression, and significantly increased levels of ICAM-1, IL-6, and TNF-α, along with enhanced monocyte adhesion. These findings reveal the damaging effects of ox-HDL on HUVECs. Transcriptomic data indicated that while SIRT1 expression did not significantly differ in coronary arteries with varying degrees of stenosis, it was notably downregulated in AS carotid artery tissues, especially in late-stage AS tissues. KEGG pathway enrichment analysis revealed that SIRT1 downregulated genes were associated with processes such as vascular smooth muscle contraction, while GO analysis showed that these downregulated genes were involved in muscle system processes and muscle contraction functions, further confirming SIRT1's critical role in AS pathology. In transcriptomic data from the SIRT1 knockout mouse model, elevated levels of inflammation-related proteins IL-6 and TNF-α were observed after SIRT1 knockout, along with decreased expression of the chaperone protein PGC-1α. The expression of mitochondrial-related functional proteins Nrf2 and PGC-1α was positively correlated with SIRT1 expression, while inflammation-related proteins ICAM-1, IL-6, IL-20, and TNF-α were negatively correlated with SIRT1 expression. We further discovered that ox-HDL triggered mitochondrial dysfunction, as evidenced by reduced expression of Mfn2, Nrf2, PGC1-α, UCP-1, and SIRT1, corroborating the results from the previous database analysis. Additionally, mitochondrial dysfunction was characterized by decreased mitochondrial membrane potential (MMP), increased mitochondrial ROS levels, and reduced ATP content, further impacting cellular energy metabolism and respiratory function. Subsequent experimental results showed that the addition of AP39 mitigated these adverse effects, as evidenced by decreased levels of ICAM-1, IL-6, and TNF-α, increased eNOS expression, reduced monocyte adhesion, increased mitochondrial H₂S content, and upregulated expression of SIRT1 protein associated with mitochondrial function, reduced ROS levels, and increased ATP content. Furthermore, validation experiments using the SIRT1 inhibitor EX527 confirmed that AP39 alleviated ox-HDL-induced endothelial cell damage and mitochondrial dysfunction by upregulating SIRT1 expression., Conclusion: Ox-HDL can induce damage and mitochondrial dysfunction in HUVECs, while AP39 inhibits ox-HDL-induced endothelial cell damage and mitochondrial dysfunction by upregulating SIRT1., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In response to hyperglycemia, endothelial cells (ECs) release exosomes with altered protein content and contribute to paracrine signalling, subsequently leading to vascular dysfunction in type 2 diabetes (T2D). High glucose reprograms DNA methylation patterns in various cell/tissue types, including ECs, resulting in pathologically relevant changes in cellular and extracellular proteome. However, DNA methylation-based proteome reprogramming in endothelial exosomes and associated pathological implications in T2D are not known. Hence, in the present study, we used Human umbilical vein endothelial cells (HUVECs), High Fat Diet (HFD) induced diabetic mice (C57BL/6) and clinical models to understand epigenetic basis of exosome proteome regulation in T2D pathogenesis . Exosomes were isolated by size exclusion chromatography and subjected to tandem mass tag (TMT) labelled quantitative proteomics and bioinformatics analysis. Immunoblotting was performed to validate exosome protein signature in clinically characterized individuals with T2D. We observed ECs cultured in high glucose and aortic ECs from HFD mouse expressed elevated DNA methyltransferase1 (DNMT1) levels. Quantitative proteomics of exosomes isolated from ECs treated with high glucose and overexpressing DNMT1 showed significant alterations in both protein levels and post translational modifications which were aligned to T2D associated vascular functions. Based on ontology and gene-function-disease interaction analysis, differentially expressed exosome proteins such as Thrombospondin1, Pentraxin3 and Cystatin C related to vascular complications were significantly increased in HUVECs treated with high glucose and HFD animals and T2D individuals with higher levels of glycated hemoglobin. These proteins were reduced upon treatment with 5-Aza-2'-deoxycytidine. Our study shows epigenetic regulation of exosome proteome in T2D associated vascular complications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Endothelial dysfunction (ED) is considered to be a major driver of the increased incidence of cardiovascular disease in primary aldosteronism (PA). The functionality of the epoxyeicosatrienoic acid (EET) pathway, involving the release of beneficial endothelium-derived lipid mediators, in PA is unknown. Evidence suggests this pathway to be disturbed in various models of experimental hypertension. We therefore assessed EET production in primary human coronary artery endothelial cells exposed to aldosterone excess and measured circulating EET in patients with PA., Methods: We used qPCR to investigate changes in the expression levels of essential genes for the synthesis and degradation of EET, calcium imaging to address the functional impact on overall endothelial function, as well as mass spectrometry to determine endothelial synthetic capacity to release EET upon stimulation. RNA-seq was performed to gain further mechanistic insights. Eicosanoid concentrations in patient's plasma were also determined by mass spectrometry., Results: Aldosterone, while eliciting proinflammatory VCAM1 expression and disturbed calcium response to acetylcholine, did not negatively affect stimulated release of endothelial EET. Likewise, no differences were observed in eicosanoid concentrations in plasma from patients with PA when compared to essential hypertensive controls. However, an inhibitor of soluble epoxide hydrolase abrogated aldosterone-mediated VCAM1 induction and led to a normalized endothelial calcium response probably by restoring expression of CHRNE., Conclusion: EET release appears intact despite aldosterone excess. Epoxide hydrolase inhibition may revert aldosterone-induced functional changes in endothelial cells. These findings indicate a potential new therapeutic principle to address ED, which should be explored in future preclinical and clinical trials., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The formation and organization of complex blood vessel networks rely on various biophysical forces, yet the mechanisms governing endothelial cell-cell interactions under different mechanical inputs are not well understood. Using the dorsal longitudinal anastomotic vessel (DLAV) in zebrafish as a model, we studied the roles of multiple biophysical inputs and cerebral cavernous malformation (CCM)-related genes in angiogenesis. Our research identifies heg1 and krit1 (ccm1) as crucial for the formation of endothelial cell-cell interfaces during anastomosis. In mutants of these genes, cell-cell interfaces are entangled with fragmented apical domains. A Heg1 live reporter demonstrated that Heg1 is dynamically involved in the oscillatory constrictions along cell-cell junctions, whilst a Myosin live reporter indicated that heg1 and krit1 mutants lack actomyosin contractility along these junctions. In wild-type embryos, the oscillatory contractile forces at junctions refine endothelial cell-cell interactions by straightening junctions and eliminating excessive cell-cell interfaces. Conversely, in the absence of junctional contractility, the cell-cell interfaces become entangled and prone to collapse in both mutants, preventing the formation of a continuous luminal space. By restoring junctional contractility via optogenetic activation of RhoA, contorted junctions are straightened and disentangled. Additionally, haemodynamic forces complement actomyosin contractile forces in resolving entangled cell-cell interfaces in both wild-type and mutant embryos. Overall, our study reveals that oscillatory contractile forces governed by Heg1 and Krit1 are essential for maintaining proper endothelial cell-cell interfaces and thus for the formation of a continuous luminal space, which is essential to generate a functional vasculature., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The prevalence of coronary heart disease (CHD) has increased significantly with the aging population worldwide. It is unclear whether ferroptosis occurs during CHD. Hence, we aimed to investigate the potential mechanisms associated with ferroptosis in CHD. Bioinformatics was used to characterize differentially expressed genes (DEGs) in CHD-related datasets (GSE21610 and GSE66360). There were 76 and 689 DEGs in the GSE21610 and GSE66360, respectively, and they predominantly associated with immune and inflammatory responses. DDX3Y, EIF1AY, KDM5D, RPS4Y1, SGK1, USP9Y, and NSG1 were intersecting DEGs of GSE21610 and GSE66360. Their expression pattern in circulating endothelial cells (ECs) derived from healthy individuals and CHD patients are consistent with the results of bioinformatics analysis, especially SGK1. In vitro, SGK1 knockdown alleviated the Erastin-induced downregulation of SLC7A11, GPX4, GSH, and GSSG, as well as the upregulation of lipid peroxidation, Fe accumulation, and mitochondrial damage in mouse aortic ECs (MAECs). Notably, SGK1 may interact with NEDD4L according to the String database. Moreover, SGK1 promoted NEDD4L and p-P65 expression in MAECs. Interestingly, the effect of SGK1 knockdown on ferroptosis in MAECs was rescued by overexpression of NEDD4L or PMA (NF-κB pathway activator). In vivo, SGK1 knockdown facilitated the recovery of body weight, blood lipids, and aortic tissue structure in CHD animal models. Furthermore, SGK1 knockdown alleviated Fe accumulation in the aorta and inactivated the NEDD4L-NF-κB pathway. In conclusion, SGK1 contributes to EC ferroptosis by regulating the NEDD4L-NF-κB pathway. SGK1 could be recognized as a therapeutic target related to ferroptosis in CHD., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diabetic foot ulcers (DFUs) represent a severe complication of diabetes mellitus. Ramulus Mori (Sangzhi) alkaloids (SZ-A), an approved oral medication for type 2 diabetes, have not been explored for their potential to enhance the processes involved in diabetic wound healing. This study aims to investigate SZ-A's role in diabetic wound healing mechanisms. The in vivo experimentation involves dividing the subjects into NC and SZ-A groups, with SZ-A dosed at 200 and 400 mg/kg, to assess the therapeutic efficacy of SZ-A. The results of the animal studies show that SZ-A intervention accelerates the processes of diabetic angiogenesis and wound healing in a manner dependent on its concentration. Additionally, a pathological model using advanced glycation end products (AGEs) in HUVECs demonstrates SZ-A's cytoprotective effect. In vitro, SZ-A intervention significantly increases cell proliferation, migration and tube formation, protecting HUVECs from oxidative stress injury induced by AGEs. Mechanistically, SZ-A exerts a protective effect on HUVECs from oxidative stress damage through the activation of the NRF2/HO-1/eNOS signaling pathway. The findings suggest that SZ-A exhibits considerable potential as a promising candidate for treating DFUs, which will aid in more effectively integrating plant-based therapies into clinical settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)