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Cell-bound membrane vesicles (CBMVs), a novel type of membrane vesicles, have been identified through a series of characterization tools. However, the lipid composition of CBMVs has not yet been characterized. This study focuses on the differences in lipid composition between CBMVs and cell membranes. In order to determine the lipid composition of CBMVs and cell membranes of Human umbilical vein endothelial cells (HUVECs) and find out differential metabolites, this study was carried out by isolating CBMVs lipids and characterizing them using high-performance liquid chromatography tandem secondary mass spectrometry (LC-MS/MS). The results showed the presence of 213 up-regulated and 726 down-regulated lipids in CBMVs compared to cell membranes which produced CBMVs. There are lipids expressed in CBMVs and not in cell membranes: DGDG 18:0_8:0, DGDG O-8:0_16:1, DGDG O-26:7_26:7, DGDG O-16:3_26:7, TG 15:4_21:5_22:5; 4O, PC 49:11, PG 19:5_38:10, PI 60:14, PI 44:9, PI 25:2, PI 43:5, PI 50:10, PS 55:10. DGDG (digalactosyl diglyceride), MGDG (monogalactosyl diglyceride) belongs to galactosyl diglyceride, promotes fat catabolism, which also has antioxidant and anti-inflammatory effects, and unsaturated diacylglycerols are a class of antioxidant compounds, which enables CBMVs to have a therapeutic potential., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract: Expression of the thrombomodulin (TM) variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). However, little is known regarding the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of the TM536 variant as well as overexpression cell models, we demonstrated that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum (ER) membrane, then, because of the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536, which is poorly modified by chondroitin sulfate glycosaminoglycan compared with conventionally secreted soluble forms of TM, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C (PC). We also showed that TM536 cellular trafficking was altered, with retention in the early secretory pathway and increased sensitivity to ER-associated degradation. As expected, activation of ER-associated degradation increased TM536 degradation and reduced its release. The expression of TM536 at the cell surface was low, and its distribution in lipid raft-like membrane microdomains was altered, resulting in low thrombin-dependent PC activation on the cell surface., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Pathological conditions such as oxidative stress or inflammation may alter the homeostasis of adventitia triggering vascular wall remodeling and abnormal angiogenesis, what can lead to development of atherosclerosis. Growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine and metabolic regulator, but its role in angiogenesis is not yet fully defined. Here we utilized an organ-on-a-chip technology to analyze endothelial sprouting in an adventitia-resembling microenvironment. We analyzed angiogenic responses to growth factor gradient across the extracellular matrix-resembling fibrin gel and in cell co-culture in response to GDF-15-treated adventitial fibroblasts. We observed that GDF-15 enhanced the pro-angiogenic effect of vascular endothelial growth factor. On the other hand, GDF-15-treated adventitial fibroblasts decreased endothelial sprouting. GDF-15 seems to indirectly affect endothelial cells and, depending on the microenvironment, its effect can be either pro- or anti-angiogenic., 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.)

Background: The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation., Methods and Results: Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system ( YAP5SA CM-SMRTs or GFP CM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SA CM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SA CM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFP CM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFP CM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SA CM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SA CM-SMRTs appeared to promote cardiomyocyte proliferation., Conclusions: The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

The imbalance of vascular endothelial cell homeostasis is the key mechanism for the progression of many vascular diseases. RNA modification, particularly N 6 -Methyladenosine (m 6 A), plays important function in numerous biological processes. Nevertheless, the regulatory function of m 6 A RNA methylation in endothelial dysfunction remains insufficiently characterized. In this study, we established that the m 6 A methyltransferase METTL3 is critical for regulating endothelial function. Functionally, depletion of METTL3 results in decreased endothelial cells proliferation, survival and inflammatory response. Conversely, overexpression of METTL3 elicited the opposite effects. Mechanistically, MeRIP-seq identified that METTL3 catalyzed m 6 A modification of TRAF1 mRNA and enhanced TRAF1 translation, thereby up-regulation of TRAF1 protein. Over-expression of TRAF1 successfully rescued the inhibition of proliferation and adhesion of endothelial cells due to METTL3 knockdown. Additionally, m 6 A methylation-mediated TRAF1 expression can be reversed by the demethylase ALKBH5. Knockdown of ALKBH5 upregulated the level of m 6 A and protein level of TRAF1, and also increased endothelial cells adhesion and inflammatory response. Collectively, our findings suggest that METTL3 regulates vascular endothelium homeostasis through TRAF1 m 6 A modification, suggesting that targeting the METTL3-m 6 A-TRAF1 axis may hold therapeutic potential for patients with 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 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: MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear., Methods and Results: Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading, and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the phosphoinositide 3-kinase/protein kinase B pathway, enhancing platelet activation through activating the integrin αIIbβ3-mediated outside-in signalling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease in platelet activity., Conclusion: Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy., 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.)

Aims: SCUBE2 (signal peptide-CUB-epidermal growth factor-like domain-containing protein 2) is a secreted or membrane-bound protein originally identified from endothelial cells (ECs). Our previous work showed that SCUBE2 forms a complex with E-cadherin and stabilizes epithelial adherens junctions (AJs) to promote epithelial phenotypes. However, it remains unclear whether SCUBE2 also interacts with vascular endothelial (VE)-cadherin and modulates EC barrier function. In this study, we investigated whether and how SCUBE2 in ECs regulates vascular barrier maintenance., Methods and Results: We showed that SCUBE2 colocalized and interacted with VE-cadherin and VE-protein tyrosine phosphatase (VE-PTP) within EC AJs. Furthermore, SCUBE2 knockdown disrupted EC AJs and increased EC permeability. Expression of EC SCUBE2 was suppressed at both mRNA and protein levels via the nuclear factor-κB signalling pathway in response to pro-inflammatory cytokines or permeability-inducing agents. In line with these findings, EC-specific deletion of Scube2 (EC-KO) in mice impaired baseline barrier function and worsened vascular leakiness of peripheral capillaries after local injection of histamine or vascular endothelial growth factor. EC-KO mice were also sensitive to pulmonary vascular hyperpermeability and leucocyte infiltration in response to acute endotoxin- or influenza virus-induced systemic inflammation. Meanwhile, EC-specific SCUBE2-overexpressing mice were protected from these effects. Molecular studies suggested that SCUBE2 acts as a scaffold molecule enabling VE-PTP to dephosphorylate VE-cadherin, which prevents VE-cadherin internalization and stabilizes EC AJs. As such, loss of SCUBE2 resulted in hyperphosphorylation of VE-cadherin at tyrosine 685, which led to its endocytosis, thus destabilizing EC AJs and reducing barrier function. All of these effects were exacerbated by inflammatory insults., Conclusion: We found that SCUBE2 contributes to vascular integrity by recruiting VE-PTP to dephosphorylate VE-cadherin and stabilize AJs, thereby promoting EC barrier function. Moreover, our data suggest that genetic overexpression or pharmacological up-regulation of SCUBE2 may help to prevent vascular leakage and oedema in inflammatory diseases., 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: Cell therapy has emerged as a revolutionary tool to repair damaged tissues by restoration of an adequate vasculature. Dental Pulp stem cells (DPSC), due to their easy biological access, ex vivo properties, and ability to support angiogenesis have been largely explored in regenerative medicine., Methods: Here, we tested the capability of Dental Pulp Stem Cell-Conditioned medium (DPSC-CM), produced in normoxic (DPSC-CM Normox) or hypoxic (DPSC-CM Hypox) conditions, to support angiogenesis via their soluble factors. CMs were characterized by a secretome protein array, then used for in vivo and in vitro experiments. In in vivo experiments, DPSC-CMs were associated to an Ultimatrix sponge and injected in nude mice. After excision, Ultimatrix were assayed by immunohistochemistry, electron microscopy and flow cytometry, to evaluate the presence of endothelial, stromal, and immune cells. For in vitro procedures, DPSC-CMs were used on human umbilical-vein endothelial cells (HUVECs), to test their effects on cell adhesion, migration, tube formation, and on their capability to recruit human CD14 + monocytes., Results: We found that DPSC-CM Hypox exert stronger pro-angiogenic activities, compared with DPSC-CM Normox, by increasing the frequency of CD31 + endothelial cells, the number of vessels and hemoglobin content in the Ultimatrix sponges. We observed that Utimatrix sponges associated with DPSC-CM Hypox or DPSC-CM Normox shared similar capability to recruit CD45 - stromal cells, CD45 + leukocytes, F4/80 + macrophages, CD80 + M1-macrophages and CD206 + M2-macropages. We also observed that DPSC-CM Hypox and DPSC-CM Normox have similar capabilities to support HUVEC adhesion, migration, induction of a pro-angiogenic gene signature and the generation of capillary-like structures, together with the ability to recruit human CD14 + monocytes., Conclusions: Our results provide evidence that DPSCs-CM, produced under hypoxic conditions, can be proposed as a tool able to support angiogenesis via macrophage polarization, suggesting its use to overcome the issues and restrictions associated with the use of staminal cells., (© 2024. The Author(s).)

Integrity of epithelial and endothelial cell barriers is of critical importance for health, barrier disruption is a hallmark of numerous diseases, of which many are driven by carbonyl stressors such as methylglyoxal (MG). Carnosine and anserine exert some MG-quenching activity, but the impact of these and of other histidine containing dipeptides on cell barrier integrity has not been explored in detail. In human proximal tubular (HK-2) and umbilical vein endothelial (HUVEC) cells, exposure to 200 µM MG decreased transepithelial resistance (TER), i.e. increased ionic permeability and permeability for 4-, 10- and 70-kDa dextran, membrane zonula occludens (ZO-1) abundance was reduced, methylglyoxal 5-hydro-5-methylimidazolones (MG-H1) formation was increased. Carnosine, balenine (ß-ala-1methyl-histidine) and anserine (ß-ala-3-methyl-histidine) ameliorated MG-induced reduction of TER in both cell types. Incubation with histidine, 1-/3-methylhistidine, but not with ß-alanine alone, restored TER, although to a lower extent than the corresponding dipeptides. Carnosine and anserine normalized transport and membrane ZO-1 abundance. Aminoguanidine, a well-described MG-quencher, did not mitigate MG-induced loss of TER. Our results show that the effects of the dipeptides on epithelial and endothelial resistance and junction function depend on the methylation status of histidine and are not exclusively explained by their quenching activity., (© 2024. The Author(s).)

Neutrophils are essential cells involved in inflammation. However, the specific mechanism of neutrophil chemotaxis induced by Treponema pallidum remains unknown. In this study, human umbilical vein endothelial cells (HUVECs) were utilized as target cells to investigate the expression levels of chemokines when stimulated with different concentrations of Tp0768 (also known as TpN44.5 or TmpA, a T. pallidum infection dependent antigen). The results indicated that Tp0768 treatment enhanced neutrophil chemotaxis in HUVECs, which was closely associated with the expression levels of CXCL1, CXCL2, and CXCL8 (also known as interleukin-8). At the same time, the results show that the Toll-like receptor 2 (TLR2) signaling pathway is activated and that endoplasmic reticulum (ER) stress occurs. Furthermore, the findings revealed that the use of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and immunoglobulin-regulated enhancer 1 (IRE1) inhibitors reduced the expression levels of CXCL1, CXCL2, and CXCL8. Additionally, inhibiting TLR2 significantly decreased the expression levels of ER stress-related proteins (PERK and IRE1), CXCL1, CXCL2, and CXCL8. Consequently, neutrophil chemotaxis was significantly inhibited after treatment with TLR2, PERK, and IRE1 inhibitors. These findings shed light on the role of Tp0768 in enhancing neutrophil chemotaxis in endothelial cells, providing a foundation for further exploration of syphilis pathogenesis and offering a new direction for the diagnosis and treatment of T. pallidum infection., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. 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.)

Periodontitis is a significant independent risk factor for atherosclerosis. Yet, the exact mechanism of action is still not fully understood. In this study, we investigated the effect of exosomes-miR-155-5p derived from periodontal endothelial cells on atherosclerosis in vitro and in vivo. Higher expression of miR-155-5p was detected in the plasma exosomes of patients with chronic periodontitis (CP) and carotid atherosclerosis (CAS) compared to patients with CP. Also, the expression level of miR-155-5p was associated with the severity of CP. miR-155-5p-enriched exosomes from HUVECs increased the angiogenesis and permeability of HAECs and promoted the expression of angiogenesis, permeability, and inflammation genes. Along with the overexpression or inhibition of miR-155-5p, the biological effect of HUVECs-derived exosomes on HAECs changed correspondingly. In ApoE-/- mouse models, miR-155-5p-enriched exosomes promoted the occurrence of carotid atherosclerosis by increasing permeable and angiogenic activity. Collectively, these findings highlight a molecular mechanism of periodontitis in CAS, uncovering exosomal miR-155-5p derived periodontitis affecting carotid endothelial cells in an 'exosomecrine' manner. Exosomal miR-155-5p may be used as a biomarker and target for clinical intervention to control this intractable disease in future, and the graphic abstract was shown in Figure S1., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Coronary artery disease (CAD) causes myocardial ischemia, narrowing or occlusion of the lumen. Although great progress has been made in the treatment of CAD, the existing treatment methods do not meet the clinical needs, so it is urgent to find new treatment methods. The aim of this study was to investigate the mechanism of action of miR-148a-3p in alleviating CAD by inhibiting vascular endothelial cell injury and to provide new ideas for the treatment of CAD. A cell model was constructed by lipopolysaccharide (LPS) induction of vascular endothelial cells, and a CAD rat model was established by a high-fat diet and intraperitoneal injection of posterior pituitary hormone. Relevant indices were detected by RT-qPCR, ELISA, Western blot, MTT, and flow cytometry. The results indicate that in LPS-induced vascular endothelial cell assays, miR-148a-3p inhibited the upregulation of PCSK9, thereby suppressing the NF-κB signaling pathway and promoting vascular endothelial cell proliferation. Overexpression of PCSK9 and the addition of NF-κB signaling pathway activator increased vascular endothelial cell apoptosis. In animal experiments, miR-148a-3p alleviated the symptoms of CAD rats, whereas overexpression of PCSK9 promoted apoptosis and increased atheromatous plaque area in CAD rats. In conclusion, miR-148a-3p inhibits the NF-κB signaling pathway through downregulation of PCSK9, thereby protecting vascular endothelial cells and alleviating CAD., (© 2024 Wiley Periodicals LLC.)

Polycyclic aromatic hydrocarbons (PAHs) exposure is associated with cardiovascular diseases. Toxic effects of PAHs are diverse, while cardiovascular consequences of benzo[b]fluoranthene (B[b]F) are unclear. Here, we reported the impacts of B[b]F on coronary artery and atherosclerosis markers both in mice and umbilical vein endothelial EAhy.926 cells. In mice, we found that B[b]F decreases heart-to-body weight ratio, affects aortic physiology, elevates serum low-density lipoprotein and total cholesterol, increases aortic levels of collagen fiber and atherosclerotic marker vascular cell adhesion molecule-1 (VCAM-1), and downregulates oxidative stress related nuclear factor erythroid 2-related factor 2 (Nrf2). In EAhy.926 cells, we showed that B[b]F inhibits cell proliferation and migration in a dose-dependent manner, induces cell cycle arrest and apoptosis, increases reactive oxygen species, upregulates VCAM-1 level, and suppresses expression of Nrf2. Taken together, our findings reveal that B[b]F exposure may contribute to coronary artery damage and potentially induce atherosclerosis, possibly via the Nrf2-related signaling pathways., Competing Interests: Declaration of Competing Interest The authors declare no conflicts., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.)

Endometriosis is a multifactorial gynecological disease, with angiogenesis as a key hallmark. The role of exosomal microRNAs (miRNAs) in endometriosis is not well understood. This study investigates differentially expressed exosomal miRNAs linked to angiogenesis in endometriosis, clarifies their molecular mechanisms, and identifies potential targets. Primary endometrial stromal cells (ESCs) were cultured, and exosomes were extracted. In a co-culture system, ESC-derived exosomes were taken up by human umbilical vein endothelial cells (HUVECs). Endometriosis implant-ESC-derived exosomes (EI-EXOs) significantly promoted HUVEC proliferation, migration and tube formation compared to normal endometrium-exosomes (NE-EXOs), a finding consistent in vivo in mice. MiRNA sequencing and bioinformatics identified differentially expressed miR-21-5p from EI-EXOs, confirmed by RT-qPCR. The miR-21-5p inhibitor or GW4869 attenuated EI-EXO-induced HUVEC proliferation, migration, and tube formation. TIMP3 overexpression diminished the pro-angiogenic effect of EI-EXOs, which was reversed by adding EI-EXOs or upregulating miR-21-5p. These findings validate the crosstalk between ESCs and HUVECs mediated by exosomal miR-21-5p, and confirm the miR-21-5p-TIMP3 axis in promoting angiogenesis in endometriosis. KEY MESSAGES: ESC-derived exosomes were found to be taken up by recipient cells, i.e. HUVECs. Functionally, endometriosis implant-ESC-derived exosomes (EI-EXOs) could significantly promote the proliferation, migration and tube formation of HUVECs compared to normal endometrium-exosomes (NE-EXOs). Through miRNA sequencing and bioinformatics analysis, differentially expressed miR-21-5p released by EI-EXOs was chosen, as confirmed by qRT-PCR. miR-21-5p inhibitor or GW4869 was found to attenuate the proliferation, migration, and tube formation of HUVECs induced by EI-EXOs. In turn, TIMP3 overexpression diminished the pro-angiogenic effect of EI-EXOs, and this angiogenic phenotype was reversed once EI-EXOs were added or miR-21-5p was upregulated., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Previous studies have highlighted the antitumor effects of mesenchymal stem cell‑derived extracellular vesicles (MSC‑EVs), positioning them as a promising therapeutic avenue for cancer treatment. However, some researchers have proposed a bidirectional influence of MSC‑EVs on tumors, determined by the specific tissue origin of the MSCs and the types of tumors involved. The present study aimed to elucidate the effects of human placenta MSC‑derived extracellular vesicles (hPMSC‑EVs) on the malignant behavior of a mouse breast cancer model of 4T1 cells in vitro and in vivo . The findings revealed that hPMSC‑EVs significantly inhibited the proliferation, migration and colony formation of cultured 4T1 mouse breast cancer cells without inducing apoptosis. Exposure to conditioned medium from 4T1 cells pretreated with hPMSC‑EVs resulted in decreased angiogenic activity, accompanied by the downregulation of angiogenesis‑promoting genes in human umbilical vein endothelial cells. In murine xenograft models derived from the 4T1 cell line, local administration of hPMSC‑EVs substantially hindered tumor growth. Further results revealed that hPMSC‑EVs inhibited angiogenesis in vivo , as reflected by the use of a vascular growth factor receptor 2‑Fluc transgenic mouse model. In summary, the results confirmed that hPMSC‑EVs negatively modulated breast cancer growth by suppressing tumor cell proliferation and migration via an indirect antiangiogenic mechanism. These results underscored the therapeutic potential of EVs, suggesting a promising avenue for alternative anticancer treatments in the future.

Peripheral nerve injury (PNI) often leads to significant functional impairment. Here, we investigated the impact of epidermal growth factor-like domain-containing protein 7 (EGFL7) on angiogenesis and nerve regeneration following PNI. Using a sciatic nerve injury model, we assessed nerve function using the sciatic nerve function index. We analyzed the expression levels of EGFL7, forkhead box proteins A1 (FOXA1), nerve growth factor (NGF), brain-derived neurotrophic factors (BDNF), Neurofilament 200 (NF200), myelin protein zero (P0), cell adhesion molecule 1 (CD31), vascular endothelial growth factor (VEGF), and NOTCH-related proteins in tissues and cells. Cell proliferation, migration, and angiogenesis were evaluated through cell counting kit assays, 5-ethynyl-2'deoxyuridine staining, and Transwell assays. We investigated the binding of FOXA1 to the EGFL7 promoter using dual-luciferase assays and chromatin immunoprecipitation. We observed decreased EGFL7 expression and increased FOXA1 expression in PNI, and EGFL7 overexpression alleviated gastrocnemius muscle atrophy, increased muscle weight, and improved motor function. Additionally, EGFL7 overexpression enhanced Schwann cell and endothelial cell proliferation and migration, promoted tube formation, and upregulated NGF, BDNF, NF200, P0, CD31, and VEGF expression. FOXA1 was found to bind to the EGFL7 promoter region, inhibiting EGFL7 expression and activating the NOTCH signaling pathway. Notably, FOXA1 overexpression counteracted the effects of EGFL7 on Schwann cells and endothelial cells. In conclusion, EGFL7 holds promise as a therapeutic molecule for treating sciatic nerve injury., (© 2024 International Federation of Cell Biology.)

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor characterized by extensive angiogenesis. However, the underlying mechanisms of HCC pathogenesis remain unclear. Previous studies have shown that RNA-binding proteins (RBPs) are implicated in HCC pathogenesis. In this study, we observed that increased RBM28 expression in HCC tissues was positively correlated with tumor microvascular density and negatively correlated with patient prognosis. Overexpression of RBM28 in HCC cells promoted tubule formation in human umbilical vein endothelial cells, whereas inhibition of RBM28 had the opposite effect, furthermore, the role of RBM28 in the progression of HCC was assessed using transgenic mouse models and chemically induced HCC models. We used various molecular assays and high-throughput detection methods to evaluate the role of RBM28 in promoting angiogenesis in HCC. Increased RBM28 expression in HCC directly binds to STAT3 mRNA, recruiting EIF4E to increase STAT3 expression and enhancing the secretion and expression of vascular endothelial growth factor A; consequently, promoting neovascularization in HCC. The potential of RBM28 as a viable diagnostic and therapeutic target for HCC was assessed using multi-cohort clinical samples and animal models. In summary, our results provide insights into the pathogenesis, clinical diagnosis, and treatment of HCC., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs)., Methods: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25-160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot., Results: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01)., Conclusions: The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis., (© 2023. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

After myocardial ischemia/reperfusion injury (MI/RI), endothelial cell injury causes impaired angiogenesis and obstruction of microcirculation, resulting in an inflammatory outburst that exacerbates the damage. Therefore, synergistic blood vessel repair and inflammation inhibition are effective therapeutic strategies. In this study, we developed a platelet membrane (PM)-encapsulated baicalin nanocrystalline (BA NC) nanoplatform with a high drug load, BA NC@PM, which co-target to endothelial cells and macrophages through the transmembrane proteins of the PM to promote angiogenesis and achieve anti-inflammatory effects. In vitro cell scratch assays and transwell assay manifested that BA NC@PM could promote endothelial cell migration, as well as increase mRNA expression of CD31 and VEGF in the heart after treatment of MI/RI mice, suggesting its favorable vascular repair function. In addition, the preparation significantly reduced the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors in plasma, promoting the polarization of macrophages. Our study highlights a strategy for enhancing the treatment of MI/RI by promoting angiogenesis and regulating macrophage polarization via the biomimetic BA NC@PM nanoplatform., 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 B.V. All rights reserved.)

Paeoniflorin (Pae) can improve diabetes mellitus (DM), especially endothelial dysfunction induced by high glucose (HG). Molecularly, the mechanism pertinent to Pae and DM lacks further in-depth research. Hence, this study determined the molecular mechanism of Pae in treating DM through network pharmacology. The target of Pae was analyzed by TCMSP database, and DM-related genes were dissected by Genecards database and Omim database. PPI network was constructed for cross targets through Cytoscape 3.9.1 and STRING platform. GO and KEGG analyses were carried out on the cross targets. Protein molecular docking verification was completed by AutoDockTools and Pymol programs. Human umbilical vein endothelial cells (HUVECs) were separately treated with HG, Pae (5, 10, 20 μM) and/or HRAS overexpression plasmids (oe-HRAS). The cell viability, apoptosis and the protein expressions of HRAS and Ras-GTP were evaluated. There were 50 cross targets between Pae and DM, and VEGFA, EGFR, HRAS, SRC and HSP90AA1 were the key genes identified by PPI network analysis. GO and KEGG analyses revealed signal paths such as Rap1 and Ras. Molecular docking results confirmed that Pae had a good binding ability with key genes. In HG-treated HUVECs, Pae dose-dependently facilitated cell viability, attenuated cell apoptosis, and dwindled the expressions of HRAS and Ras-GTP, but these effects of Pae were reversed by oe-HRAS. In conclusion, Pae regulates the viability and apoptosis of HG-treated HUVECs by inhibiting the expression of HRAS.

5-Fluorouracil (5-FU) is a first-line treatment for colorectal cancer, but side effects such as severe diarrhea are common in clinical use and have been linked to its induction of normal cell senescence. Chloramphenicol (CAP) is an antibiotic commonly used to treat typhoid or anaerobic infections, but its senescence-related aspects have not been thoroughly investigated. Here, we used 5-FU to induce senescence in human umbilical vein endothelial cells (HUVECs) and investigated the relationship between CAP and cellular senescence at the cellular level. In a model of cellular senescence induced by 5-FU treatment, we discovered that CAP treatment reversed the rise in the percentage of senescence-associated galactosidase (SA-β-gal)-positive cells and decreased the expression of senescence-associated proteins (p16), senescence-associated genes (p21), and senescence-associated secretory phenotypes (SASPs: IL-6, TNF-α). In addition, CAP subsequently restored the autophagic process inhibited by 5-FU and upregulated the levels of autophagy-related proteins. Mechanistically, we found that CAP restored autophagic flux by inhibiting the mTOR pathway, which in turn alleviated FU-induced cellular senescence. Our findings suggest that CAP may help prevent cellular senescence and restore autophagy, opening up new possibilities and approaches for the clinical management of colorectal cancer., Competing Interests: The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Liver fibrosis is a chronic pathological process in which the abnormal proliferation of connective tissue is induced by various pathogenic factors. During the process of fibrosis, excessive angiogenesis is observed. Physiological angiogenesis has the potential to impede the progression of liver fibrosis through augmenting matrix metalloenzyme activity; however, pathological angiogenesis can exacerbate liver fibrosis by promoting collagen accumulation. Therefore, a key scientific research focus in the treatment of liver diseases is to search for the "on-off" mechanism that regulates angiogenesis from normal proliferation to pathological proliferation. In this study, we found that excessive angiogenesis appeared during the initial phase of hepatic fibrosis without mesenchymal characteristics. In addition, angiogenesis accompanied by significant endothelial-to-mesenchymal transition (EndMT) was observed in mice after the intraperitoneal injection of angiotensin II (Ang II). Interestingly, the changes in Yes-associated protein (YAP) activity in endothelial cells (ECs) can affect the regulation of angiogenesis by Ang II. The results of in vitro experiments revealed that the regulatory influence of Ang II on ECs was significantly attenuated upon suppression of YAP activity. Furthermore, the function of Ang II in regulating angiogenesis during fibrosis was investigated in liver-specific transgenic mice. The results revealed that Ang II gene deletion could restrain liver fibrosis and EndMT. Meanwhile, Ang II deletion downregulated the profibrotic YAP signaling pathway in ECs. The small molecule AT1R agonist olmesartan targeting Ang II-YAP signaling could also alleviate liver fibrosis. In conclusion, this study identified Ang II as a pivotal regulator of EndMT during the progression of liver fibrosis and evaluated the therapeutic effect of the Ang II-targeted drug olmesartan on liver fibrosis., 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 Inc. All rights reserved.)

Background & Aims: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS., Methods: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data., Results: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS., Conclusions: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: The 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor (TF) procoagulant activity (PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu 98 -Leu 115 epitope in GRP78., Objectives: Determine the effects of the engagement of the anti-GRP78 autoantibody to csGRP78 on ECs and the underlying mechanisms that impact TF PCA., Methods: Immunofluorescent staining was used to determine the presence of csGRP78 in tumor necrosis factor α-treated ECs. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay (Abcam) was used to quantify changes in intracellular Ca 2+ levels. Small molecules predicted to bind GRP78 were identified using artificial intelligence. Enzyme-linked immunosorbent assays were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex., Results: In tumor necrosis factor α-treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by endoplasmic reticulum stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca 2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin attenuated anti-GRP78 autoantibody-induced TF PCA. We identified B07 ∗ , as a GRP78 binder that diminished anti-GRP78 autoantibody-induced TF PCA on ECs., Conclusion: These findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Diabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells., Objectives: We investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia., Methods: Immunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA., Results: Platelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H 2 O 2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors., Conclusion: Endothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

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

To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis., Competing Interests: Conflict of interest The authors have no conflicts to disclose., (Copyright © 2024. Published by Elsevier B.V.)

The alteration of inflammatory phenotype by macrophage polarization plays an important role in diabetic wound repair. Apigenin has been reported to be anti-inflammatory and promote tissue repair; however, whether it regulates macrophage polarization to participate in diabetic wound repair remains to be investigated. We found that apigenin promoted miR-21 expression in LPS-stimulated RAW264.7 cells, inhibited cellular M1-type factor TNF-α and IL-1β secretion and increased M2-type factor IL-10 and TGF-β secretion, and accelerated macrophage conversion from M1 type to M2 type, whereas this protective effect of apigenin was counteracted by a miR-21 inhibitor. Moreover, we established a macrophage-HUVECs cell in vitro co-culture system and found that apigenin accelerated the migration, proliferation, and VEGF secretion of HUVECs by promoting macrophage miR-21 expression. Further, mechanistic studies revealed that this was mediated by the TLR4/Myd88/NF-κB axis. In in vivo study, diabetic mice had significantly delayed wound healing compared to non-diabetic mice, accelerated wound healing in apigenin-treated diabetic mice, and decreased M1-type macrophages and increased M2-type macrophages in wound tissues., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Renal fibrosis serves as the shared pathway in chronic kidney disease (CKD) progression towards end-stage renal disease (ESRD). Endothelial-mesenchymal transition (EndMT) is a vital mechanism leading to the generation of myofibroblasts, thereby contributing to the advancement of fibrogenesis. Baculoviral IAP Repeat Containing 3(Birc3) was identified as a crucial inhibitor of cell death and a significant mediator in inflammatory signaling and immunity. However, its involvement in the development of renal interstitial fibrosis via EndMT still needs to be clarified. Herein, elevated levels of Birc3 expression along with EndMT-associated alterations, including increased α-smooth muscle actin (α-SMA) levels and decreased CD31 expression, were observed in fibrotic kidneys of Unilateral Ureteral Obstruction (UUO)-induced mouse models and transforming growth factor-β (TGF-β)-induced EndMT in Human Umbilical Vein Endothelial Cells (HUVECs). Functionally, Birc3 knockdown inhibited EndMT and mitochondrial fission mediated by dynamin-related protein 1 (Drp1) both in vivo and in vitro. Mechanistically, endothelial Birc3 exacerbated Drp-1-induced mitochondrial fission through the MAPK/PI3K/Akt signaling pathway in endothelial cell models stimulated TGF-β. Collectively, these findings illuminate the mechanisms and indicate that targeting Birc3 could offer a promising therapeutic strategy to improve endothelial cell survival and mitigate the progression of CKD., 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 Inc.)

Vascular endothelial senescence is a major risk factor for diabetic vascular complications. Abnormal mitochondrial fission by dynamically related protein 1 (DRP1) accelerates vascular endothelial cell senescence. Homoplantaginin (Hom) is a flavonoid in Salvia plebeia R. Br. with protecting mitochondrial and repairing vascular properties. However, the relevant mechanism of Hom against diabetic vascular endothelial cell senescence remains unclear. Here, we used db/db mice and high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs) to assess the anti-vascular endothelial cell senescence of Hom. We found that Hom inhibited senescence-associated β-galactosidase activity, decreased the levels of senescence markers, and senescence-associated secretory phenotype factors. Additionally, Hom inhibited the expression of cGAS-STING pathway and downstream inflammatory factors. STING inhibitor H-151 delayed endothelial senescence, whereas STING overexpression attenuated the anti-endothelial senescence effect of Hom. Furthermore, we observed that Hom reduced mitochondrial fragmentation and inhibited abnormal mitochondrial fission using transmission electron microscopy. Importantly, Hom has a stronger effect on mitochondrial fission protein than mitochondrial fusion protein, especially downregulated the expression of DRP1. DRP1 inhibitor Mdivi-1 suppressed cGAS-STING pathway and vascular endothelial senescence, yet DRP1 agonist FCCP attenuated the effect of Hom. Surprisingly, Hom blunted abnormal mitochondrial fission mediated by DRP1 mitochondrial localization, suppressed interaction of DRP1 with VDAC1 and prevented VDAC1 oligomerization, which was necessary for mtDNA escape and subsequent cGAS-STING pathway activation. These results revealed a previously unrecognized mechanism that Hom alleviated vascular endothelial senescence by inhibited mtDNA-cGAS-STING signaling pathway via blunting DRP1-mitochondrial fission-VDAC1 axis., (© 2024 Federation of American Societies for Experimental Biology.)

Vascular endothelial cytoskeletal disruption leads to increased vascular permeability and is involved in the pathogenesis and progression of various diseases. Oxidative stress can increase vascular permeability by weakening endothelial cell-to-cell junctions and decrease intracellular nicotinamide adenine dinucleotide (NAD + ) levels. However, it remains unclear how intracellular NAD + variations caused by oxidative stress alter the vascular endothelial cytoskeletal organization. In this study, we demonstrated that oxidative stress activates poly (ADP-ribose [ADPr]) polymerase (PARP), which consume large amounts of intracellular NAD + , leading to cytoskeletal disruption in vascular endothelial cells. We found that hydrogen peroxide (H 2 O 2 ) could transiently disrupt the cytoskeleton and reduce intracellular total NAD levels in human umbilical vein endothelial cells (HUVECs). H 2 O 2 stimulation led to rapid increase in ADPr protein levels in HUVECs. Pharmaceutical PARP inhibition counteracted H 2 O 2 -induced total NAD depletion and cytoskeletal disruption, suggesting that NAD + consumption by PARP induced cytoskeletal disruption. Additionally, supplementation with nicotinamide mononucleotide (NMN), the NAD + precursor, prevented both intracellular total NAD depletion and cytoskeletal disruption induced by H 2 O 2 in HUVECs. Inhibition of the NAD + salvage pathway by FK866, a nicotinamide phosphoribosyltransferase inhibitor, maintained H 2 O 2 -induced cytoskeletal disruption, suggesting that intracellular NAD + plays a crucial role in recovery from cytoskeletal disruption. Our findings provide further insights into the potential application of PARP inhibition and NMN supplementation for the treatment and prevention of diseases involving vascular hyperpermeability., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Atherosclerotic cardiovascular diseases remain the leading cause of mortality in diabetic patients, with endothelial cell (EC) dysfunction serving as the initiating step of atherosclerosis, which is exacerbated in diabetes. Krüppel-like factor 11 (KLF11), known for its missense mutations leading to the development of diabetes in humans, has also been identified as a novel protector of vascular homeostasis. However, its role in diabetic atherosclerosis remains unexplored., Methods: Diabetic atherosclerosis was induced in both EC-specific KLF11 transgenic and knockout mice in the Ldlr -/- background by feeding a diabetogenic diet with cholesterol (DDC). Single-cell RNA sequencing (scRNA-seq) was utilized to profile EC dysfunction in diabetic atherosclerosis. Additionally, gain- and loss-of-function experiments were conducted to investigate the role of KLF11 in hyperglycemia-induced endothelial cell dysfunction., Results: We found that endothelial KLF11 deficiency significantly accelerates atherogenesis under diabetic conditions, whereas KLF11 overexpression remarkably inhibits it. scRNA-seq profiling demonstrates that loss of KLF11 increases endothelial-to-mesenchymal transition (EndMT) during atherogenesis under diabetic conditions. Utilizing gain- and loss-of-function approaches, our in vitro study reveals that KLF11 significantly inhibits EC inflammatory activation and TXNIP-induced EC oxidative stress, as well as Notch1/Snail-mediated EndMT under high glucose exposure., Conclusion: Our study demonstrates that endothelial KLF11 is an endogenous protective factor against diabetic atherosclerosis. These findings indicate that manipulating KLF11 could be a promising approach for developing novel therapies for diabetes-related cardiovascular complications., (© 2024. The Author(s).)

Abstract: Loss of endothelial barrier function contributes to the pathophysiology of many inflammatory diseases. Coagulation factor XI (FXI) plays a regulatory role in inflammation. Although activation of FXI increases vascular permeability in vivo, the mechanism by which FXI or its activated form FXIa disrupts endothelial barrier function is unknown. We investigated the role of FXIa in human umbilical vein endothelial cell (HUVEC) or human aortic endothelial cell (HAEC) permeability. The expression patterns of vascular endothelial (VE)-cadherin and other proteins of interest were examined by western blot or immunofluorescence. Endothelial cell permeability was analyzed by Transwell assay. We demonstrate that FXIa increases endothelial cell permeability by inducing cleavage of the VE-cadherin extracellular domain, releasing a soluble fragment. The activation of a disintegrin and metalloproteinase 10 (ADAM10) mediates the FXIa-dependent cleavage of VE-cadherin, because adding an ADAM10 inhibitor prevented the cleavage of VE-cadherin induced by FXIa. The binding of FXIa with plasminogen activator inhibitor 1 and very low-density lipoprotein receptor on HUVEC or HAEC surfaces activates vascular endothelial growth receptor factor 2 (VEGFR2). The activation of VEGFR2 triggers the mitogen-activated protein kinase (MAPK) signaling pathway and promotes the expression of active ADAM10 on the cell surface. In a pilot experiment using an established baboon model of sepsis, the inhibition of FXI activation significantly decreased the levels of soluble VE-cadherin to preserve barrier function. This study reveals a novel pathway by which FXIa regulates vascular permeability. The effect of FXIa on barrier function may be another way by which FXIa contributes to the development of inflammatory diseases., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Endothelial dysfunction, which is marked by a reduction in nitric oxide (NO) production or an imbalance in relaxing and contracting factor levels, exacerbates atherosclerosis by promoting the production of cell adhesion molecules and cytokines. This study aimed to investigate the effects of Limosilactobacillus reuteri HY7503, a novel probiotic isolated from raw milk, on endothelial dysfunction. Five lactic acid bacterial strains were screened for their antioxidant, anti-inflammatory, and endothelium-protective properties; L. reuteri HY7503 had the most potent effect. In a mouse model of angiotensin II-induced endothelial dysfunction, L. reuteri HY7503 reduced vascular thickening (19.78%), increased serum NO levels (226.70%), upregulated endothelial NO synthase ( eNOS ) expression in the aortic tissue, and decreased levels of cell adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and serum cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin-6 [IL-6]). In TNF-α-treated human umbilical vein endothelial cells (HUVECs), L. reuteri HY7503 enhanced NO production and reduced cell adhesion molecule levels. In HUVECs, surface-layer proteins (SLPs) were more effective than extracellular vesicles (exosomes) in increasing NO production and decreasing cell adhesion molecule levels. These findings suggested that L. reuteri HY7503 may serve as a functional probiotic that alleviates endothelial dysfunction.

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in the migration of leukocytes or cancer cells through the endothelium during inflammation or metastasis. Migrating cells exert forces on the endothelium through cell surface adhesion molecules, such as platelet endothelial adhesion molecule PECAM-1, and this is essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads and exerted about 40 pN force on the endothelial monolayer. We show that force increases cell-ECM adhesion in the cell center and is accompanied by the opening of cell-cell junctions. Upon depletion of the MEK/ERK kinase, BRAF force increases cell-ECM adhesion both at the cell periphery and in the cell center, but this does not result in the opening of cell-cell junctions. Decreasing cell-ECM adhesion in BRAF-depleted cells through FAK inhibition results in the remodeling of cell-cell junctions. Force-induced increase in cell-ECM adhesion in the cell center correlates with the activation of the transcriptional cofactor Yes-associated protein (YAP). Furthermore, the induced activation of YAP through LATS inhibition prevents junctional remodeling in control cells. Thus, the activation of YAP might determine the strength of cell-cell junctions during PECAM-1-mediated mechanotransduction.

Metabolic memory (MM) is a major factor in the delayed wound healing observed in diabetic patients. While "Nocardia rubrum cell wall skeleton" (Nr-CWS) is utilized to enhance macrophage proliferation in immune diseases, its impact on MM wounds in diabetes is unclear. This study demonstrates that transient hyperglycemia leads to prolonged damage in vascular endothelial cells by decreasing METTL3 expression, leading to decreased RNA methylation and impaired cellular metabolism. Remarkably, Nr-CWS application increases METTL3 levels in these cells, facilitating the recovery of cell function. Further in vivo and in vitro analyses demonstrate that transient hyperglycemia-induced reduction in METTL3 hinders RNA methylation of the downstream gene Cds2, impacting mitochondrial function and energy metabolism and consequently reducing angiogenic capacity in endothelial cells. This impairment significantly influences diabetic wound healing. Our findings highlight the profound impact of transient hyperglycemia on wound healing, establishing METTL3 as a significant role in vascular complications of diabetes. This study not only elucidates the pathophysiological mechanisms behind MM in diabetic wounds but also suggests Nr-CWS as a potential therapeutic agent, offering a novel approach for treating diabetic wounds., (© 2024. The Author(s).)

Contrast-induced acute kidney injury (CIAKI) is a common complication with limited treatments. Intermedin (IMD), a peptide belonging to the calcitonin gene-related peptide family, promotes vasodilation and endothelial stability, but its role in mitigating CIAKI remains unexplored. This study investigates the protective effects of IMD in CIAKI, focusing on its mechanisms, particularly the cAMP/Rac1 signaling pathway. Human umbilical vein endothelial cells (HUVECs) were treated with iohexol to simulate kidney injury in vitro. The protective effects of IMD were assessed using CCK8 assay, flow cytometry, ELISA, and Western blotting. A CIAKI rat model was utilized to evaluate renal peritubular capillary endothelial cell injury and renal function through histopathology, immunohistochemistry, immunofluorescence, Western blotting, and transmission electron microscopy. In vitro, IMD significantly enhanced HUVEC viability and mitigated iohexol-induced toxicity by preserving intercellular adhesion junctions and activating the cAMP/Rac1 pathway, with Rac1 inhibition attenuating these protective effects. In vivo, CIAKI caused severe damage to peritubular capillary endothelial cell junctions, impairing renal function. IMD treatment markedly improved renal function, an effect negated by Rac1 inhibition. IMD protects against renal injury in CIAKI by activating the cAMP/Rac1 pathway, preserving peritubular capillary endothelial integrity and alleviating acute renal injury from contrast media. These findings suggest that IMD has therapeutic potential in CIAKI and highlight the cAMP/Rac1 pathway as a promising target for preventing contrast-induced acute kidney injury in at-risk patients, ultimately improving clinical outcomes.

Atherosclerosis is a leading cause of cardiovascular diseases, characterized by endothelial dysfunction and lipid accumulation. Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell behavior. This study aimed to investigate the role of lncRNA NORAD in endothelial cell proliferation and as a potential therapeutic target for atherosclerosis. A total of 75 CAD patients and 76 controls were recruited, and plasma NORAD levels were measured using qRT-PCR. HUVECs were transfected with si-NORAD to evaluate its effects on cell cycle, proliferation, migration, and apoptosis. Plasma NORAD levels were significantly elevated in CAD patients. The NORAD-miRNA-mRNA ceRNA regulatory network was constructed based on GEO database, and G1/S-specific cyclin-D1 (CCND1) was identified as one of the hub factors. NORAD deficiency suppressed cell migration and induced G1 cell cycle arrest in HUVECs by downregulating CCND1 in vitro. NORAD upregulated CCND1 in HUVECs via sponging miR-106a that inhibited cell migration. The dual-luciferase assay confirmed the direct targeting of miR-106a by NORAD, and overexpression of miR-106a inhibited HUVEC proliferation and migration. Si-NORAD transfection resulted in induced early apoptosis, increased intracellular ROS levels, decreased GSH levels, and reduced mitochondrial membrane potential. Additionally, si-NORAD decreased the expression of GPX4, FTH1, KEAP1, NCOA4, and Nrf2, while increasing Xct levels, confirming the involvement of ferroptosis. Our findings reveal that NORAD plays a critical role in endothelial cell proliferation, migration, and apoptosis, and its silencing induces ferroptosis. The regulatory network involving NORAD, miR-106a, and their target genes provides a potential therapeutic avenue for atherosclerosis., (© 2024. The Author(s).)

Glaucine is an aporphine alkaloid with anti-inflammatory, bronchodilator and anti-cancer activities. However, the effects of glaucine in the regulation of age-related macular degeneration (AMD) remain unclear. Herein, we aimed to investigate the anti-angiogenetic and anti-inflammatory effects of glaucine in ARPE-19 cells. ARPE-19 cells were treated with N-(methoxyoxoacetyl)-glycine, methyl ester (DMOG) and cobalt chloride (CoCl 2 ) for induction of hypoxia, while lipopolysaccharide (LPS) treatment was used for elicitation of inflammatory response. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The expression of hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by Western blot. The secretion of VEGF, interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was detected using enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were used for tube formation analysis. Expression of HIF-1α and secretion of VEGF were significantly increased under DMOG and CoCl 2 induction, whereas glaucine significantly attenuated both HIF-1α expression and VEGF secretion by DMOG- and CoCl 2 -induced ARPE-19 cells. In addition, glaucine suppressed the tube formation by DMOG- and CoCl 2 -induced HUVEC cells. Moreover, glaucine also attenuated the production of IL-6 and MCP-1 by LPS-induced ARPE-19 cells. This study indicated that glaucine exhibited anti-angiogenic and anti-inflammatory effects, suggesting that glaucine might have benefits for the treatment of AMD., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cadmium is a contributing factor to cardiovascular diseases and highly toxic to vascular endothelial cells. It has a distinct mode of injury, causing the de-endothelialization of regions in the monolayer structure of endothelial cells in a concentration-dependent manner. However, the specific molecules involved in the cadmium toxicity of endothelial cells remain unclear. The purpose of this study was to identify the specific molecular mechanisms through which cadmium affects endothelial detachment. Cadmium inhibited the expression of claudin-5 and zonula occludens (ZO)-1, which are components of tight junctions (strongest contributors to intercellular adhesion), in a concentration- and time-dependent manner. Compared to arsenite, zinc, and manganese, only cadmium suppressed the expression of both claudin-5 and ZO-1 molecules. Moreover, the knockdown of claudin-5 and ZO-1 exacerbated cadmium-induced endothelial cell injury and expansion of the detachment area, whereas their overexpression reversed these effects. CRE-binding protein inhibition reduced cadmium toxicity, suggesting that CRE-binding protein activation is involved in the cadmium-induced inhibition of claudin-5 and ZO-1 expression and endothelial detachment. These findings provide new insights into the toxicological mechanisms of cadmium-induced endothelial injury and risk of cardiovascular disease.

Background: Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications., Methods: The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined., Result: Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model., Conclusion: Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds., (© 2024. The Author(s).)

Aims: Endothelial cell (EC) dysfunction plays a key role in the initiation and progression of cardiovascular disease. However, studying these disorders in ECs from patients is challenging; hence, the use of human induced pluripotent stem cells (hiPSCs) and their in vitro differentiation into ECs represents a very promising approach. Still, the generation of hiPSC-derived ECs (hECs) remains demanding as a cocktail of growth factors and an intermediate purification step are required for hEC enrichment. Therefore, we probed the utility of a forward programming approach using transgenic hiPSC lines., Methods and Results: We have used the transgenic hiPSC line PGP1 ETV2 isoform 2 to explore the in vitro differentiation of hECs via doxycycline-dependent induction of the ETS variant transcription factor 2 (ETV2) and compared these with a standard differentiation protocol for hECs using non-transgenic control hiPSCs. The transgenic hECs were highly enriched without an intermediate purification step and expressed-as non-transgenic hECs and human umbilical vein endothelial cells-characteristic EC markers. The viability and yield of transgenic hECs were strongly improved by applying EC growth medium during differentiation. This protocol was successfully applied in two more transgenic hiPSC lines yielding reproducible results with low line-to-line variability. Transgenic hECs displayed typical functional properties, such as tube formation and LDL uptake, and a more mature phenotype than non-transgenic hECs. Transgenic hiPSCs preferentially differentiated into the arterial lineage; this was further enhanced by adding a high concentration of vascular endothelial growth factor to the medium. We also demonstrate that complexing lentivirus with magnetic nanoparticles and application of a magnetic field enables efficient transduction of transgenic hECs., Conclusion: We have established a highly efficient, cost-effective, and reproducible differentiation protocol for the generation of functional hECs via forward programming. The transgenic hECs can be genetically modified and are a powerful tool for disease modelling, tissue engineering, and translational purposes., 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.)

Endothelial cell (EC) senescence and vascular aging are important hallmarks of chronic metabolic diseases. An improved understanding of the precise regulation of EC senescence may provide novel therapeutic strategies for EC and vascular aging-related diseases. This study examined the potential functions of Spinster homolog 2 (SPNS2) in EC senescence and vascular aging. We discovered that the expression of SPNS2 was significantly lower in older adults, aged mice, hydrogen peroxide-induced EC senescence models and EC replicative senescence model, and was correlated with the expression of aging-related factors. in vivo experiments showed that the EC-specific knockout of SPNS2 markedly aggravated vascular aging by substantially, impairing vascular structure and function, as evidenced by the abnormal expression of aging factors, increased inflammation, reduced blood flow, pathological vessel dilation, and elevated collagen levels in a naturally aging mouse model. Moreover, RNA sequencing and molecular biology analyses revealed that the loss of SPNS2 in ECs increased cellular senescence biomarkers, aggravated the senescence-associated secretory phenotype (SASP), and inhibited cell proliferation. Mechanistically, silencing SPNS2 disrupts pyruvate metabolism homeostasis via pyruvate kinase M (PKM), resulting in mitochondrial dysfunction and EC senescence. Overall, SPNS2 expression and its functions in the mitochondria are crucial regulators of EC senescence and vascular aging., (© 2024. The Author(s).)

Vascular endothelial damage due to ionizing radiation is the main pathological process of radiation injury and the main cause of damage to various organs in nuclear accidents. Ferroptosis plays an important role in ionizing radiation-induced cell death. We have previously reported that luteolin is highly resistant to ferroptosis. In the present study, body weight, microvessel count, H&E, and Masson staining results showed that luteolin rescued radial vascular injury in vivo. Cell Counting Kit 8 (CCK8), Giemsa staining clarified the anti-ferroptosis ability of luteolin with low toxicity. Malondialdehyde (MDA), superoxide dismutase (SOD), NADP+/NADPH, Fe2+ staining, dihydroethidium (DHE) and MitoTracker assays for ferroptosis-related metrics, we found that luteolin enhances human umbilical vein endothelial cells (HUVECs) antioxidant damage capacity. Drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), computer simulated docking and western blot showed that heat shock protein beta-1 (HSPB1) is one of the targets of luteolin action. Luteolin inhibits ferroptosis by promoting the protein expression of HSPB1/solute carrier family 7 member 11 (SLC7A11)/ glutathione peroxidase 4 (GPX4). In conclusion, we have preliminarily elucidated the antioxidant damage ferroptosis ability and the target of action of luteolin to provide a theoretical basis for the application of luteolin in radiation injury diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Studies have found that miRNAs can participate in the progression of hypertension by affecting the function of endothelial cells and inflammatory response. This study was to investigate the clinical value of miR-320b in patients with hypertension and its potential effect on Angiotensin (Ang) II-induced endothelial cells. Real-time quantitative PCR (RT-qPCR) was used to detect the differential expression of miR-320b in all subjects, and the diagnostic value of miR-320b in hypertension was further evaluated by the receiver operating characteristic (ROC) curve. Ang II-induced human umbilical vein endothelial cells (HUVECs) were established as a model of hypertension injury. The possible downstream target gene AKT serine/threonine kinase 3 (AKT) of miR-320b was predicted through TargetScan, and the interaction between miR-320b and AKT3 was verified by luciferase reporter gene. The results showed that serum miR-320b was reduced in patients with hypertension compared with healthy people (P < 0.001). With the increase of hypertension grade, the serum miR-320b level of patients gradually decreased (P < 0.001). ROC analysis showed that miR-320b had the ability to distinguish patients from healthy people. Cell analysis proved that Ang II induced the decrease of HUVECs viability and the activation of apoptosis and inflammation, while overexpression of miR-320b inhibited Ang II-induced apoptosis and inflammation and promoted cell growth (P < 0.05). Luciferase reporter gene showed that AKT3 was the downstream target gene of miR-320b. In summary, this study suggests that miR-320b alleviates Ang II-induced apoptosis, inflammation and the inhibition of cell viability by targeting AKT3 expression, and may be involved in the pathogenesis of hypertension.

Pleiotrophin (PTN) is a secreted factor that regulates endothelial cell migration through protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) and α v β 3 integrin. Genetic deletion of Ptprz1 results in enhanced endothelial cell proliferation and migration, due to the decreased expression of β 3 integrin and the subsequent, enhanced cMet phosphorylation. In the present study, we investigated the effect of PTN and PTPRZ1 on activating the mTORC1 kinase and protein synthesis and identified part of the implicated signaling pathway in endothelial cells. PTN or genetic deletion of Ptprz1 activates protein synthesis in a mTORC1-dependent manner, as shown by the enhanced phosphorylation of the mTORC1-downstream targets ribosomal protein S6 kinase 1 (SK61) and 4E-binding protein 1 (4EBP1) and the upregulation of HIF-1α. The cMet tyrosine kinase inhibitor crizotinib abolishes the stimulatory effects of PTN or PTPRZ1 deletion on mTORC1 activation and protein synthesis, suggesting that mTORC1 activation is downstream of cMet. The mTORC1 inhibitor rapamycin abolishes the stimulatory effect of PTN or PTPRZ1 deletion on endothelial cell migration, suggesting that mTORC1 is involved in the PTN/PTPRZ1-dependent cell migration. The α v β 3 integrin blocking antibody LM609 and the peptide PTN 112-136 , both known to bind to α ν β 3 and inhibit PTN-induced endothelial cell migration, increase cMet phosphorylation and activate mTORC1, suggesting that cMet and mTORC1 activation are required but are not sufficient to stimulate cell migration. Overall, our data highlight novel aspects of the signaling pathway downstream of the PTN/PTPRZ1 axis that regulates endothelial cell functions.