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Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated flow chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676-2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027-3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716-5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general., 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 Ltd. All rights reserved.)

Objectives: To explore the effect of exosomal miR-126 derived from stem cells from the apical papilla (SCAPs) under hypoxia on human umbilical vein endothelial cell (HUVEC) angiogenesis., Methods: miR-126 mimics plasmids were used to upregulate miR-126 in SCAPs. Internalization of PKH26-labeled exosomes was examined by fluorescent microscopy. CCK-8 assay, Transwell assay, scratch assay, tube formation assay, and Matrigel plug assay were performed to detect the effects of exosomes on the angiogenic ability of HUVECs. The luciferase reporter assay and rescue assay were performed to examine the relationship between miR-126 and sprouty-related, EVH1 domain-containing protein 1 (SPRED1). The involvement of SPRED1 and the extracellular signal-regulated kinase (ERK) signaling pathway was evaluated by western blotting., Results: miR-126 expression was upregulated in SCAPs and in SCAP-derived exosomes under hypoxia. miR-126 expression was increased in HUVECs when cocultured with SCAP-derived exosomes. Induced overexpression of miR-126 in hypoxic SCAPs and secreted exosomes resulted in enhanced angiogenesis both in vitro and in vivo. Western blot analysis revealed that miR-126-mediated SPRED1 downregulation induced activation of ERK signaling., Conclusions: Under hypoxic conditions, exosomes derived from SCAPs can promote HUVEC angiogenesis through expression of miR-126, which subsequently suppresses SPRED1 and activates the ERK signaling pathway., (© 2022 Wiley Periodicals LLC.)

Introduction: Pregnancy is a dynamic time period associated with significant physiological changes in the cardiovascular system. It is well known that during pregnancy, the placenta secretes a variety of molecular signals, including exosomes, into the maternal circulation to adapt to increased blood volume and maintain blood pressure at normotensive levels., Methods: In the present study, we compared the effects of exosomes derived from the peripheral blood serum of nonpregnant women (NP-Exo) and pregnant women with uncomplicated pregnancy (P-Exo) on endothelial cell function. We also analyzed the proteomic profiles of these two groups of exosomes and the molecular mechanisms underlying the effect of exosome cargoes on vascular endothelial cell function., Results: We found that P-Exo were positively involved in regulating the function of human umbilical vein endothelial cell (HUVEC) and promoting the release of nitric oxide (NO). Furthermore, we revealed that trophoblast-derived pregnancy-specific beta-1-glycoprotein 1 (PSG1)-enriched exosomes treatment induced the promotion of HUVEC proliferation and migration as well as the release of NO. In addition, we found that P-Exo maintained blood pressure at normal levels in mice., Discussion: These results suggested that PSG1-enriched exosomes derived from maternal peripheral blood regulate the function of vascular endothelial cells and play an important role in maintaining maternal blood pressure during pregnancy., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Angiogenesis is essential for cardiac repair after myocardial infarction. Promoting angiogenesis has been demonstrated as an effective approach for myocardial infarction treatment. Several different strategies for inducing myocardial angiogenesis have been explored, including exogenous delivery of angiogenic genes, proteins, microRNAs, cells, and extracellular vesicles. Various types of injectable hydrogels have been investigated for cardiac tissue repair. One of the most promising injectable hydrogels in cardiac regeneration is a cardiac extracellular matrix hydrogel that is derived from decellularized porcine myocardium. It can be delivered minimally invasively via transendocardial delivery. The safety and efficacy of cardiac extracellular matrix hydrogels have been shown in small and large animal myocardial infarction models as well as clinical trials. The main mechanisms underlying the therapeutic benefits of cardiac extracellular matrix hydrogels have been elucidated and involved in the modulation of the immune response, downregulation of pathways related to heart failure progression and fibrosis, upregulation of genes important for cardiac muscle contraction, and enhancing cardiomyocyte differentiation and maturation from stem cells. However, no potent capillary network formation induced by cardiac extracellular matrix hydrogels has been reported. In this study, we tested the feasibility of incorporating a fibrin matrix into cardiac extracellular matrix hydrogels to improve the angiogenic properties of the hydrogel. Our in vitro results demonstrate that fibrin-enriched cardiac extracellular matrix hydrogels can induce robust endothelial cell tube formation from human umbilical vein endothelial cells and promote the sprouting of human mesenchymal stem cell spheroids. The obtained information from this study is very critical toward the future in vivo evaluation of fibrin-enriched cardiac extracellular matrix hydrogels in promoting myocardial angiogenesis.

Wound healing is a highly orchestrated process involving many cell types, such as keratinocytes, fibroblasts and endothelial cells. This study aimed to evaluate the potential application of synthetic peptides derived from tilapia piscidin (TP)2, TP2-5 and TP2-6 in skin wound healing. The treatment of HaCaT keratinocytes with TP2-5 and TP2-6 did not cause cytotoxicity, but did enhance cell proliferation and migration, which could be attributed to the activation of epidermal growth factor receptor signaling. In CCD-966SK fibroblasts, although TP2-5 (31.25 μg/mL) and TP2-6 (125 μg/mL) showed cytotoxic effects, we observed the significant promotion of cell proliferation and migration at low concentrations. In addition, collagen I, collagen III, and keratinocyte growth factor were upregulated by the peptides. We further found that TP2-5 and TP2-6 showed pro-angiogenic properties, including the enhancement of human umbilical vein endothelial cell (HUVEC) migration and the promotion of neovascularization. In a murine model, wounds treated topically with TP2-5 and TP2-6 were reduced by day 2 post-injury and healed significantly faster than untreated wounds. Taken together, these findings demonstrate that both TP2-5 and TP2-6 have multifaceted effects when used as topical agents for accelerating wound healing.

Endothelial cell apoptosis is an important pathophysiology in many cardiovascular diseases. The gasotransmitter nitric oxide (NO) is known to regulate cell survival and apoptosis. However, the mechanism underlying the effect of NO remains unclear. In this research, by targeting cytosolic copper/zinc superoxide dismutase (SOD1) monomerization, we aimed to explore how NO inhibited endothelial cell apoptosis. We showed that treatment with the NO synthase (NOS) inhibitor nomega-nitro-l-arginine methyl ester hydrochloride (L-NAME) significantly decreased the endogenous NO content of endothelial cells, facilitated the formation of SOD1 monomers, inhibited dismutase activity, and promoted reactive oxygen species (ROS) accumulation in human umbilical vein endothelial cells (HUVECs); by contrast, supplementation with the NO donor sodium nitroprusside (SNP) upregulated NO content, prevented the formation of SOD1 monomers, enhanced dismutase activity, and reduced ROS accumulation in L-NAME-treated HUVECs. Mechanistically, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a specific reducer of cysteine thiol, increased SOD1 monomer formation, thus preventing the NO-induced increase in dismutase activity and the decrease in ROS. Furthermore, SNP inhibited HUVEC apoptosis caused by the decrease in endogenous NO, whereas TCEP abolished this protective effect of SNP. In summary, our data reveal that NO protects endothelial cells against apoptosis by inhibiting cysteine-dependent SOD1 monomerization to enhance SOD1 activity and inhibit oxidative stress., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A. membranaceus is a traditional Chinese medicine that regulates blood sugar levels, suppresses inflammation, protects the liver, and enhances immunity. In addition, A. membranaceus is also widely used in diet therapy and is a well-known health tonic. Formononetin is a natural product isolated from A. membranaceus that has multiple biological functions, including anti-cancer activity. However, the mechanism by which formononetin inhibits tumor growth is not fully understood. In this present study, we demonstrated that formononetin suppresses PD-L1 protein synthesis via reduction of MYC and STAT3 protein expression. Furthermore, formononetin markedly reduced the expression of MYC protein via the RAS/ERK signaling pathway and inhibited STAT3 activation through JAK1/STAT3 pathway. Co-immunoprecipitation experiments illustrated that formononetin suppresses protein expression of PD-L1 by interfering with the interaction between MYC and STAT3. Meanwhile, formononetin promoted PD-L1 protein degradation via TFEB and TFE3-mediated lysosome biogenesis. T cell killing assay revealed that formononetin could enhance the activity of cytotoxic T lymphocytes (CTLs) and restore ability to kill tumor cells in a co-culture system of T cells and tumor cells. In addition, formononetin inhibited cell proliferation, tube formation, cell migration, and promoted tumor cell apoptosis by suppressing PD-L1. Finally, the inhibitory effect of formononetin on tumor growth was confirmed in a murine xenograft model. The present study revealed the anti-tumor potential of formononetin, and the findings should support further research and development of anti-cancer drugs for cervical cancer., (Copyright © 2021. Published by Elsevier Inc.)

Endothelial cell dysfunction is an essential pathophysiological feature of preeclampsia (PE). It has been reported that cathepsin C is upregulated in the maternal vascular endothelium of PE patients. The excessive activation of p38 MAPK leads to various diseases, including PE. NF-κB pathway can promote uteroplacental dysfunction, endothelial stress and development of PE. Moreover, it has been verified that cathepsin C can activate p38 MAPK/NF-κB pathway. In the present work, hypoxia/reoxygenation (H/R) injury model of HUVECs was established to discuss the biological functions of cathepsin C in endothelial cell dysfunction and to elucidate the underlying molecular mechanism. The correlation between cathepsin C and p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs as well as the effects of cathepsin C and p38 MAPK/NF-κB pathway on viability, apoptosis, invasion, in vitro angiogenesis of HUVECs and oxidative stress were assessed. The results revealed that H/R injury elevated cathepsin C expression and activated p38 MAPK/NF-κB pathway in HUVECs and cathepsin C knockdown inhibited the activity of p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs. Downregulation of cathepsin C improved viability, inhibited apoptosis and enhanced invasion of H/R-stimulated HUVECs. In addition, downregulation of cathepsin C alleviated oxidative stress and induced stronger HUVEC angiogenesis in vitro. Furthermore, the protective effects of cathepsin C knockdown against endothelial cell dysfunction were reversed by p38 MAPK activator anisomycin. In other words, downregulation of cathepsin C could improve HUVEC viability and enhance anti-apoptotic capacity, anti-oxidative capability, invasive ability, as well as angiogenic potential of H/R-stimulated HUVECs by repressing p38 MAPK/NF-κB pathway.

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m 6 A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m 6 A methyltransferase. Through m 6 A sequencing and functional studies, we determined that m 6 A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m 6 A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m 6 A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation., Competing Interests: BL, TZ, ML, ZC, YZ, ML, YL, YS, ML, YT, YY, HJ, DL No competing interests declared, (© 2022, Li et al.)

Bioactive glass (BG) has recently shown great promise in soft tissue repair, especially in wound healing; however, the underlying mechanism remains unclear. Pyroptosis is a novel type of programmed cell death that is involved in various traumatic injury diseases. Here, we hypothesized that BG may promote wound healing through suppression of pyroptosis. To test this scenario, we investigated the possible effect of BG on pyroptosis in wound healing both in vivo and in vitro. This study showed that BG can accelerate wound closure, granulation formation, collagen deposition, and angiogenesis. Moreover, western blot analysis and immunofluorescence staining revealed that BG inhibited the expression of pyroptosis-related proteins in vivo and in vitro. In addition, while BG regulated the expression of connexin43 (Cx43), it inhibited reactive oxygen species (ROS) production. Cx43 activation and inhibition experiments further indicate that BG inhibited pyroptosis in endothelial cells by decreasing Cx43 expression and ROS levels. Taken together, these studies suggest that BG promotes wound healing by inhibiting pyroptosis via Cx43/ROS signaling pathway., (© 2021. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Osteoclastogenesis in alveolar bone induced by compression stress triggers orthodontic tooth movement. Compression stress also stimulates angiogenesis, which is essential for osteoclastogenesis. However, the effects of osteoclastogenesis induced by compression on angiogenesis are poorly understood. In vivo, we found the markers of angiogenesis increased during orthodontic bone remodeling. In vitro, osteoclast-derived exosomes increased proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs), as well as expression of vascular endothelial growth factor and CD31. The promotive effects of exosomes derived from compressed osteoclasts were greater than those derived from osteoclasts without compression. Next, we analyzed changes in the microRNA transcriptome after compression stress and focused on microRNA146a-5p (miR-146a), which was significantly decreased by compression. Transfection of an inhibitor of miR-146a stimulated angiogenesis of HUVECs while miR-146a mimics repressed angiogenesis. Adiponectin (ADP) was confirmed to be a target of miR-146a by dual luciferase reporter assay. In HUVECs treated with exosomes, we detected increased ADP which promoted angiogenesis. Knockdown of ADP in HUVECs reduced the promotive effects of exosomes. Our results demonstrate that the decreased miR-146a observed in osteoclasts after compression promotes angiogenesis by targeting ADP, suggesting a novel method to interfere with bone remodeling induced by compression stress., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5'-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5'-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

Background: C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences., Methods: Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10  μ g/mL, 24 hours), (3) CRP receptor (Fc γ RIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm 2 , 24 hours), and (5) HiLSS followed by CRP stimulation., Results: AA HUVECs had significantly higher Fc γ RIIB receptor expression under both basal and CRP incubation conditions. Blocking Fc γ RIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects., Conclusion: Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on Fc γ RIIB receptor as a potential contributor to racial differences in endothelial function in AA., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Chenyi Ling et al.)

Whether alterations in the microtubule cytoskeleton affect the ability of endothelial cells (ECs) to sprout and form branching networks of tubes was investigated in this study. Bioassays of human EC tubulogenesis, where both sprouting behavior and lumen formation can be rigorously evaluated, were used to demonstrate that addition of the microtubule-stabilizing drugs, paclitaxel, docetaxel, ixabepilone, and epothilone B, completely interferes with EC tip cells and sprouting behavior, while allowing for EC lumen formation. In bioassays mimicking vasculogenesis using single or aggregated ECs, these drugs induce ring-like lumens from single cells or cyst-like spherical lumens from multicellular aggregates with no evidence of EC sprouting behavior. Remarkably, treatment of these cultures with a low dose of the microtubule-destabilizing drug, vinblastine, led to an identical result, with complete blockade of EC sprouting, but allowing for EC lumen formation. Administration of paclitaxel in vivo markedly interfered with angiogenic sprouting behavior in developing mouse retina, providing corroboration. These findings reveal novel biological activities for pharmacologic agents that are widely utilized in multidrug chemotherapeutic regimens for the treatment of human malignant cancers. Overall, this work demonstrates that manipulation of microtubule stability selectively interferes with the ability of ECs to sprout, a necessary step to initiate and form branched capillary tube networks., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor β1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. We hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

Background: YKL-40, a secreted glycoprotein, has a role in promoting tumor angiogenesis through syndecan-1 receptor. Syndecan-4 is a member of syndecan family. However, the effects of YKL-40 on migration and tube formation of human umbilical vein cells (HUVECs) mediated by syndecan-4 receptor are unknown. Materials and methods: HUVECs were transfected with lentivirus encoding syndecan-4 short hairpin (sh) RNAs (lenti-synd4 shRNAs) and the efficiency of transfection was measured using qRT-PCR and western blotting. The effects of recombinant protein of YKL-40 on migration and angiogenesis of HUVECs adjusted by syndecan-4 were determined by wound healing and tube formation assay. The expressions of protein kinase Cα (PKCα) and extracellular signal regulated kinases (ERKs) 1 and 2 (ERK1/2) in HUVECs were measured using western blotting. Results: The mRNA and protein expression of syndecan-4 were significantly decreased in HUVECs successfully transfected with lenti-synd4 shRNAs. Lenti-synd4 shRNAs remarkably inhibited the migration and tube formation of HUVECs stimulated by recombinant protein of YKL-40. The levels of PKCα and ratio of p-ERK1/2 to ERK1/2 in HUVECs were also decreased by down-regulating syndecan-4. Conclusion: The effects of YKL-40 on migration and tube formation of HUVECs are partly inhibited by knock-downing syndecan-4 through suppressing PKCα and ERK1/2 signaling pathways., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose : To assess the anti-neovascularization effect of a novel peptide NT/K-CFY derived from the kringle domain of neurotrypsin. Materials and Methods : Cell migration, lumen formation and cell proliferation assays were performed to determine the anti-neovascularization effect of NT/K-CFY in primary human umbilical vein endothelial cells (HUVECs). Chick chorioallantoic membrane (CAM) and oxygen-induced retinopathy (OIR) models were established to assess the anti-angiogenic role of NT/K-CFY in vivo . The retinal expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) was examined by western blot and real-time PCR in OIR model. Results : The in vitro results showed that NT/K-CFY effectively and safely decreased VEGF-induced cell migration, cell proliferation and tube formation in HUVECs. In addition, NT/K-CFY showed certain efficacy in angiogenesis inhibition in chicken embryos and oxygen-treated mouse pups. Moreover, the CFY peptide also improved retinal blood perfusion and reversed the abnormal expression of VEGF and PEDF in OIR mouse model. Conclusion : NT/K-CFY peptide strongly inhibits neovascularization in vitro and vivo . This novel peptide may become a promising therapeutic agent for ocular angiogenesis-related diseases.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO 2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Gestational diabetes mellitus (GDM) affects one in four Saudi women and is associated with high risks of cardiovascular diseases in both the mother and foetus. It is believed that endothelial cells (ECs) dysfunction initiates these diabetic complications. In this study, differences in the transcriptome profiles, cell cycle distribution, and mitochondrial superoxide (MTS) between human umbilical vein endothelial cells (HUVECs) from GDM patients and those from healthy (control) subjects were analysed. Transcriptome profiles were generated using high-density expression microarray. The selected four altered genes were validated using qRT-PCR. MTS and cell cycle were analysed by flow cytometry. A total of 84 altered genes were identified, comprising 52 upregulated and 32 downregulated genes in GDM.HUVECs. Our selection of the four interested altered genes (TGFB2, KITLG, NEK7, and IGFBP5) was based on the functional network analysis, which revealed that these altered genes are belonging to the highest enrichment score associated with cellular function and proliferation; all of which may contribute to ECs dysfunction. The cell cycle revealed an increased percentage of cells in the G2/M phase in GDM.HUVECs, indicating cell cycle arrest. In addition, we found that GDM.HUVECs had increased MTS generation. In conclusion, GDM induces persistent impairment of the biological functions of foetal ECs, as evidenced by analyses of transcriptome profiles, cell cycle, and MTS even after ECs culture in vitro for several passages under normal glucose conditions.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor β1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study aimed to explore the angiogenesis potential of human endothelial cells encapsulated inside alginate-gelatin microspheres under static and dynamic culture systems after 7 days. Human umbilical vein endothelial cells were encapsulated inside alginate (1%) and gelatin (1.2%) using an electrostatic encapsulation method. Cells were incubated for 7 days in vitro. The cell survival rate was measured using the MTT assay. The expression of VEGFR-2 and von Willebrand factor genes was studied by real-time PCR assay. Using western blot analysis, we monitored the protein contents of VEGFR-2, vWF, and Caspase 3. The levels of SOD and GPx enzymes were calculated using biochemical kits. Angiogenesis potential was assessed using in vitro Matrigel assay. Data showed an increased survival rate in encapsulated cells cultured under the static condition compared to the conventional 2D condition (p < 0.05). The culture of encapsulated cells under a dynamic bioreactor system did not alter cell viability. Compared to the dynamic culture system, the incubation of encapsulated cells in the static culture system swelled the microspheres (p < 0.05). Both dynamic and static culture models increased the expression of VEGFR-2 and von Willebrand factor in encapsulated cells compared to 2D culture (p < 0.05), showing enhanced functional maturation. Data showed a significant increase of vWF and reduction of apoptosis marker Caspase in the dynamic culture system (p < 0.05). The levels of SOD and GPx were significantly increased in dynamic and static culture models as compared to the control 2D group (p < 0.05). In vitro tubulogenesis assay showed significant induction of angiogenesis in dynamic encapsulated HUVECs indicated with a large number of vascular tubes and arborized ECs compared to the control and static encapsulated HUVECs (p < 0.05). The current study suggests a bioreactor dynamic system is a reliable approach, similar to a static condition, for the expansion of encapsulated human ECs in a 3D milieu., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exosomes secreted by adipose-derived stem cells (ADSCs) enhance angiogenesis and wound healing. However, in clinical settings, wounds may be infected by various bacteria or pathogens. We investigated whether human ADSCs stimulated with lipopolysaccharide (LPS) secrete exosomes (ADSC-LPS-exo) that augment the angiogenesis of human umbilical vein endothelial cells (HUVECs). ExoQuick-TC exosome precipitation solution was used to purify exosomes from human ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h. The uptake of ADSC-LPS-exo significantly induced the activation of cAMP response element binding protein (CREB), activating protein 1 (AP-1), and nuclear factor-κB (NF-κB) signaling pathways and increased the migration of and tube formation in HUVECs. RNA interference with CREB, AP-1, or NF-κB1 significantly reduced the migration of and tube formation in HUVECs treated with ADSC-LPS-exo. An experiment with an antibody array for 25 angiogenesis-related proteins revealed that only interleukin-8 expression was significantly upregulated in HUVECs treated with ADSC-LPS-exo. In addition, proteomic analysis revealed that eukaryotic translation initiation factor 4E, amyloid beta A4 protein, integrin beta-1, and ras-related C3 botulinum toxin substrate 1 may be potential candidates involved in ADSC-LPS-exo-mediated enhanced angiogenesis.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFβ2 and IL1β. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

Mitsugumin 53 (MG53), which is expressed predominantly in striated muscle, has been demonstrated to be a myokine/cardiokine secreted from striated muscle under specific conditions. The important roles of MG53 in non-striated muscle tissues have also been examined in multiple disease models. However, no previous study has implicated MG53 in the control of endothelial cell function. In order to explore the effects of MG53 on endothelial cells, human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant human MG53 (rhMG53). Then, rhMG53 uptake, focal adhesion kinase (FAK)/Src/Akt/ERK1/2 signalling pathway activation, cell migration and tube formation were determined in vitro. The efficacy of rhMG53 in regulating angiogenesis was also detected in postnatal mouse retinas. The results demonstrated that rhMG53 directly entered into endothelial cells in a cholesterol-dependent manner. The uptake of rhMG53 directly bound to FAK in endothelial cells, which resulted in a significant decrease in FAK phosphorylation at Y397. Accompanied by the dephosphorylation of FAK, rhMG53 uncoupled FAK-Src interaction and reduced the phosphorylation of Src at Y416. Consequently, the activation of FAK/Src downstream signalling pathways, such as Akt and ERK1/2, was also significantly inhibited by rhMG53. Furthermore, rhMG53 remarkably decreased HUVEC migration and tube formation in vitro and postnatal mouse retinal angiogenesis in vivo. Taken together, these data indicate that rhMG53 inhibits angiogenesis through regulating FAK/Src/Akt/ERK1/2 signalling pathways. This may provide a novel molecular mechanism for the impaired angiogenesis in ischaemic diseases., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E 2 (PGE 2 ), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

We studied the effect of nitinol, the most prevalent material for endovascular stents, on metabolic and coagulation activity of a primary culture of human umbilical vein endothelial cells (HUVEC). Metabolic activity was evaluated using MTS-test and by the level of stable NO metabolites in the conditioned medium, coagulation activity was assessed by activity of von Willebrand factor (vWF) and levels of plasminogen activator inhibitor-1 (PAI-1) and soluble endothelial protein C receptors (sEPCR). Exposure to nitinol reduced metabolic activity of the cell culture by 11.1% in comparison with the control (p<0.001). Although absolute activity of vWF and absolute level of sEPCR were elevated, incubation with nitinol did not lead to a statistically significant elevation of these parameters in comparison with the control, which can indicate the absence of substantial hypercoagulation effects of nitinol., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Adiponectin and leptin are two abundant adipokines with different properties but both described such as potent factors regulating angiogenesis. AdipoRon is a small-molecule that, binding to AdipoRs receptors, acts as an adiponectin agonist. Here, we investigated the effects of AdipoRon and leptin on viability, migration and tube formation on a human in vitro model, the human umbilical vein endothelial cells (HUVEC) focusing on the expression of the main endothelial angiogenic factors: hypoxia-inducible factor 1-alpha (HIF-1α), C-X-C motif chemokine ligand 1 (CXCL1), vascular endothelial growth factor A (VEGF-A), matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9). Treatments with VEGF-A were used as positive control. Our data revealed that, at 24 h treatment, proliferation of HUVEC endothelial cells was not influenced by AdipoRon or leptin administration; after 48 h longer exposure time, the viability was negatively influenced by AdipoRon while leptin treatment and the combination of AdipoRon+leptin produced no effects. In addition, AdipoRon induced a significant increase in complete tubular structures together with induction of cell migration while, on the contrary, leptin did not induce tube formation and inhibited cell migration; interestingly, the co-treatment with both AdipoRon and leptin determined a significant decrease of the tubular structures and cell migration indicating that leptin antagonizes AdipoRon effects. Finally, we found that the effects induced by AdipoRon administration are accompanied by an increase in the expression of CXCL1, VEGF-A, MMP-2 and MMP-9. In conclusion, our data sustain the active role of adiponectin and leptin in linking adipose tissue with the vascular endothelium encouraging the further deepening of the role of adipokines in new vessel's formation, to candidate them as therapeutic targets.

Background: Osteonecrosis of the femoral head (ONFH) is a common disease. Transplantation of bone marrow stem cells (BMSCs) is a promising method to treat ONFH but is impeded by the low survival rate and deficiency of cell bioactivity., Methods: We performed hypoxic preprocessing to treat BMSCs and assessed cell viability, apoptosis, differentiation, and growth factor expression in vitro. Subsequently, we constructed the ONFH model and delivered hypoxia-pretreated BMSCs to the rabbit femoral head after core decompression surgery, evaluating its effects on bone regeneration and ONFH repair. Six weeks later, micro-computed tomography (CT) and histopathology were performed to evaluate ONFH repair., Results: Our findings demonstrated that hypoxic preprocessing promoted the viability of BMSCs, increased the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP), calcium deposition, and enhanced the formation of vessels-shaped structures. In an in vivo study, micro-CT observations demonstrated that the bone volume was increased in the hypoxia BMSCs group. Histological examination revealed reduced cellular apoptosis, lower empty lacunae rate, enhanced bone formation, and stronger trabecular bone in the hypoxia BMSCs group when compared with those transplanted with normoxia treated BMSCs. Additionally, immunological assessment of the hypoxia BMSCs group demonstrated increased expression of HIF-1α and β-catenin, as well as increased VEGF, ALP, osteocalcin (OCN), and collagen type I (Col-1)., Conclusions: Collectively, our findings indicated that hypoxia stimulated angiogenesis and bone regeneration via the HIF-1/β-catenin pathway in BMSCs and that the delivery of hypoxia-pretreated BMSCs contributed to the treatment of early ONFH., Competing Interests: Declaration of Competing Interest We declare that there is no conflict of interest associated with this manuscript., (Copyright © 2021 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

12-LOX plays an important role in the progression of various malignancies. However, the underlying mechanisms of the action of 12-LOX and tumour treatment strategies remain not fully defined. In this study, we investigated the possible roles of 12-LOX in ESCC and explored the new therapeutic target. Approximately 73% of ESCC tissues showed marked up-regulation of 12-LOX, which was associated with poor prognosis. 12-LOX overexpression was positively correlated with the malignant progression of ESCC as demonstrated both in vitro and in vivo. Up-regulation of 12-LOX significantly increased the proliferation of ESCC cells and the xenograft volume. Moreover, 12-LOX up-regulation promoted tube formation of HUVECs and tumour angiogenesis in xenografts. Mechanism investigation indicated that 12-LOX overexpression led to activation of the PI3K/AKT/mTOR pathway and the up-regulation of VEGF in ESCC cells. Subsequent analysis indicated that the RAD001 could reverse the 12-LOX-induced promoting effect on ESCC. Specifically, the application of RAD001 inhibited the proliferation of ESCC cells and the tube-forming ability of HUVECs. In the drug group, the xenografts exhibited significant volume reduction and angiogenesis inhibition. We demonstrated that RAD001 could inhibit HUVEC migration. These findings presented the evidence that RAD001 had distinct roles on HUVECs and could exert anti-tumour effects by targeting not only the PI3K/AKT/mTOR pathway but the angiogenesis in ESCC., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Endothelial cells, due to heterogeneity in the cell structure, can potentially form an inhomogeneous on structural and mechanical properties of the inner layer of the capillaries. Using quantitative nanomechanical mapping mode of atomic force microscopy, the parameters of the structural, elastic, and adhesive properties of the cell surface for living and glutaraldehyde-fixed human umbilical vein endothelial cells were studied. A significant difference in the studied parameters for three cell surface zones (peripheral, perinuclear, and nuclear zones) was established. The perinuclear zone appeared to be the softest zone of the endothelial cell surface. The heterogeneity of the endothelial cell mechanical properties at the nanoscale level can be an important mechanism in regulating the endothelium functions in blood vessels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Knowledge regarding the relationship between the molecular mechanisms underlying atherosclerosis (AS) and transfer RNA-derived small RNAs (tsRNAs) is limited. This study illustrated the expression profile of tsRNAs, thus exploring its roles in AS pathogenesis. Small RNA sequencing was performed with four atherosclerotic arterial and four healthy subject samples. Using bioinformatics, the protein-protein interaction network and cellular experiments were constructed to predict the enriched signalling pathways and regulatory roles of tsRNAs in AS. Of the total 315 tsRNAs identified to be dysregulated in the AS group, 131 and 184 were up-regulated and down-regulated, respectively. Interestingly, the pathway of the differentiated expression of tsRNAs in cell adhesion molecules (CAMs) was implicated to be closely associated with AS. Particularly, tRF-Gly-GCC might participate in AS pathogenesis via regulating cell adhesion, proliferation, migration and phenotypic transformation in HUVECs and VSMCs. In conclusion, tsRNAs might help understand the molecular mechanisms of AS better. tRF-Gly-GCC may be a promising target for suppressing abnormal vessels functions, suggesting a novel strategy for preventing the progression of atherosclerosis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Accumulating evidence has been found that circular RNA (circRNA) plays a critical role in the initiation and development of various diseases by modulating gene expression in the cytoplasm. However, the role of circ&#95;0044366 (termed circ29) in gastric cancer (GC) has yet to be elusive. We detected that exosomal circ29 was confirmed to be highly expressed in GC and can significantly impair the proliferation, migration, tube formation of HUVEC by exosomal communication. Interestingly, this effect could be blocked by the effect of miR-29a. In brief, we confirmed that circ29, as a sponge of miR-29a, plays a responsible role in the occurrence and development of GC by regulating the VEGF pathway. Therefore, it may be used as a potential target for the treatment of GC., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The extracellular matrix (ECM) modulates a multitude of cell functions, and this regulation is provided by key ECM components forming a complex network. Hyaluronic acid (HA) is an abundant component of the ECM that binds to proteins and influences various activities of endothelial cells (ECs). Although the effect of soluble HA on cell spreading has been studied, the impact of peptide-bound HA has not yet been investigated in great detail. We aim to comprehensively study the roles of immobilized HA on the regulation of EC behavior compared to the more conventional use of soluble HA. A 2D model surface formed by self-assembled monolayers (SAMs) of a HA-binding peptide (Pep-1) is used as an anchor for HA immobilization. Mixed SAMs, consisting of thiolated Pep-1 and 1-octanethiol, are prepared and characterized by using ellipsometry and contact angle measurement. Full density Pep-1 SAMs are more hydrophilic and bind more HA than mixed SAMs. Cell spreading and migration are enhanced by immobilized low molecular weight (LMW) HA, which also facilitates cell alignment and elongation under laminar flow conditions and potentially drives directional migration. This effect is not mediated by the expression of CD44, and immobilized LMW HA is found to accelerate the assembly of focal adhesions. Such biomimetic surfaces provide new insights into the role of HA in regulating the spreading and phenotype of endothelial cells.

Problem: Intrauterine adhesion (IUA) is a uterine disorder with partial or total obstruction of the uterine cavity and/or the cervical canal primarily caused by intrauterine operations and infections. It is the most common cause of uterine infertility and recurrent abortion. However, the reasons for endometrium repair disorders in patients with IUA are still unclear. While increasing evidence demonstrates that endometrial mesenchymal stem/stromal cells (EMSCs) contribute to the regeneration and repair of endometrium, the roles of EMSCs in the pathogenesis of IUA have not been reported., Methods and Study: We investigated the differences of phenotype and biological characteristics between EMSCs from women with IUA and healthy women. Firstly, the fibrosis of endometrium were measured by immunohistochemistry and Masson staining. Second, we used immunofluorescence to detect the location of EMSCs in endometrial tissue, and the proportion of CD146 + CD140b + in the two groups was compared by flow cytometry. Then, plate colony formation experiment, CCK-8 assay, flow cytometry, would-healing assay, and transwell invasion experiment were used to compare the cloning ability, proliferation, cell cycle, migration and invasion capabilities respectively. Finally, we compared the potential angiogenesis and immunosuppression capabilities., Results: Our results showed that there were fewer CD146 + CD140b + cells in patients with IUA, and the clone-forming, migration, invasion, angiogenic and immunosuppressive abilities of the EMSCs of patients with IUA were significantly decreased compared with those of healthy women., Conclusion: There are some differences between the EMSCs of IUA patients and healthy women, which may be related to the occurrence of IUA and dysfunction of endometrium., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease., (© 2021 Federation of American Societies for Experimental Biology.)

The meshes for hernia repair result in many problems that are related to complications including chronic pain and limited movement due to inadequate mechanical strength, non-absorbability, or low elasticity. In this study, degradable polylactic acid (PLA), synthetic thermoplastic polyurethane (TPU), and acellular dermal matrix (ADM) powders are combined to prepare a novel PLA/TPU/ADM mesh with three different topological structures (square, circular, and diamond) by 3D printing. The physicochemical properties and structural characteristics of mesh are studied, the results show that the diamond structure mesh with the pore size of 3 mm has sufficient elasticity and tensile strength, which provides the efficient mechanical strength required for hernia repair (16 N cm -1 ) and the value more than polypropylene(PP) mesh. Besides, in vitro and in vivo experiments demonstrate human umbilical vein endothelial cells could successfully proliferate on the PLA/TPU/ADM mesh whose biocompatibility with the host is shown using a rat model of abdominal wall defect. In conclusion, the results of this study demonstrate that the PLA/TPU/ADM mesh may be considered a good choice for hernia repair as its potential to overcome the elastic and strength challenges associated with a highly flexible abdominal wall, as well as its good biocompatibility., (© 2021 Wiley-VCH GmbH.)

Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR‑216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF‑κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng , and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR‑216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR‑216a and the dissociation constant was 17.6  µ M. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR‑216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre‑miR‑216a recombinant lentiviruses (Lv‑miR‑216a) and the number of population‑doubling level (PDL) was calculated. Stable overexpression of miR‑216a induced a premature senescent‑like phenotype, whereas the senescent features and increased activity of senescence‑associated β‑galactosidase (SA‑β‑gal) were reversed after Rb2 treatment. The percentage of SA‑β‑gal‑positive cells in senescent PDL25 cells transfected with Lv‑miR‑216a was decreased 76% by Rb2 treatment compared with the Lv‑miR‑216a group without Rb2 treatment (P=0.01). Mechanistically, miR‑216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF‑κB‑responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro‑inflammatory effects of miR‑216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR‑216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti‑inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR‑216a.

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking in vivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10 ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4 cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72 h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design in vitro pre-vascularized tissue and graft integration prospects., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.

We studied the mechanisms by which microRNA-126 regulates proliferation and migration of human umbilical vein endothelial cells (HUVEC) cultured in a medium with high glucose concentration and treated with chemokine CXCL8. Cell proliferation, apoptosis, and migration were analyzed by the CCK-8 assay, Annexin V-PI staining, and Transwell assay, respectively. The ratios of p-ERK/ERK, p-P38/P38, p-JNK/JNK were determined by ELISA. HUVEC cells cultured in the presence of high glucose concentration (30 mmol/ml) and treated with CXCL8 (50 ng/ml) demonstrated more intensive proliferation, migration, and p-ERK/ERK, p-P38/P38, and p-JNK/JNK ratios and significantly lower apoptosis rate than control cells (high glucose, no treatment) and cells treated with CXCL8 and transfected with microRNA-126-mimic. Thus, microRNA-126 regulates proliferation and migration of HUVEC cells cultured in the presence of high glucose concentrations and treated with CXCL8 through inhibition of MAPK signaling pathway.

Context: Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear., Objective: This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis., Methods: Human Eu and Ec endometrium-derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis., Results: In total, 105 ESCs-sEV-associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference-present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis., Conclusion: The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A 2 (PLA 2 s) from snake venom have been described to possess antiangiogenic properties. In this study, we evaluated both in vitro and ex vivo antiangiogenic effects induced by BnSP-7, a Lys49 PLA 2 isolated from Bothrops pauloensis snake venom. BnSP-7 was able to inhibit endothelial cell (HUVEC) proliferation, which was indeed confirmed by a modulation of cell cycle progression. Interestingly, BnSP-7 also inhibited the adhesion and migration of HUVECs and blocked in vitro angiogenesis in a VEGF-dependent manner, an important proangiogenic factor. Finally, BnSP-7 was capable of inhibiting sprouting angiogenic process through an ex vivo aortic ring assay. Taken together, these results indicate that BnSP-7 has potent in vitro and ex vivo antiangiogenic effect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm 2 versus 20 dyn/cm 2 ). High shear stress of 20 dyn/cm 2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial joints. Inflammation, new blood vessel formation (angiogenesis) and bone resorption (osteoclastogenesis) are three key processes involved in the joint damage and deformities of arthritis. Various gut microbiota-derived metabolites are implicated in RA pathogenesis. However, there is barely any information about the impact of two such metabolites, indole-3-aldehyde (IAld) and indole-3-acetic acid (I3AA), on arthritis-related processes. We conducted a comparative analysis of IAld and I3AA using established cell-based models to understand how they might influence RA pathogenesis. Although structurally similar, the bioactivities of these two metabolites were profoundly different. IAld but not I3AA, inhibited the expression of pro-inflammatory cytokines (IL-1β and IL-6) in RAW 264.7 (RAW) cells stimulated with heat-killed M. tuberculosis sonicate (Mtb) and lipopolysaccharide (LPS). IAld also exhibited pro-angiogenic activity and pro-osteoclastogenic activity. In contrast, I3AA exhibited anti-angiogenic activity on endothelial cell tube formation but had no effect on osteoclastogenesis. Both IAld and I3AA have been proposed as aryl hydrocarbon receptor (AhR) agonists. Use of CH-223191, an inhibitor of the AhR, suppressed the anti-angiogenic activity of I3AA but failed to mitigate the effects of IAld. Further investigation of the anti-inflammatory activities of IAld and I3AA in LPS-treated RAW cells indicated that inhibition of MyD88-dependent activation of NF-κB and MAPK pathways was not likely involved. Our results suggest that the relative bioavailability of these indole derivatives may differentially impact RA progression and possibly other diseases that share similar cellular processes.

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3 , HOXA11 , and HOXA13 , and differentially expressed isoforms, such as CXCL6 and HGF . Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.

Delayed endothelial healing after drug eluting stent (DES) implantation is a critical clinical problem in treatment of coronary artery diseases. Exosomes exhibit proangiogenic potential in a variety of ischemic diseases. However, the association of exosomes with endothelial regeneration after DES implantation has been rarely reported. In this study, we aimed to investigate the therapeutic effects of mesenchymal stem cell (MSC)-derived exosomes on endothelial cells treated with rapamycin and explore the potential mechanisms of MSC-derived exosomes in promoting endothelial regeneration. Exosomes were isolated from MSCs by ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot assay. The in vitro effects of MSC-derived exosomes on the proliferation and migration of endothelial cells treated with rapamycin were evaluated by integrated experiment, cell counting kit-8, scratch, tube formation, and transwell assays. And the apoptosis of rapamycin-induced endothelial cells loaded with MSC-derived exosomes was detected using TUNEL and Annexin-V FITC and PI double-staining assays. The microRNA (miRNA) cargo of MSC-derived exosomes was identified by high-throughput RNA sequencing. Pro-angiogenic miRNAs and key pathways were further characterized. Our results indicated that MSC-derived exosomes could be ingested into umbilical vein endothelial cells (HUVECs) and significantly enhanced cell proliferation rate, migratory and tube-forming capabilities in vitro. MSC-derived exosomes also inhibited the apoptosis of HUVECs induced by rapamycin. A distinct class of exosomal miRNAs was further identified, including six miRNAs tightly related to neovasculogenesis. Silencing the expression of exosomal miRNA-21-5p and let-7c-5p attenuated the pro-proliferative and pro-migratory capacity of MSC-derived exosomes. Moreover, functional enrichment analysis indicated that metabolic pathways might contribute to reendothelialization. This study highlights a proregenerative effect of MSC-derived exosomes in vitro, which may be partly explained by the delivery of pro-angiogenic miRNAs to endothelial cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC-derived cardiomyocytes., (© 2020 Wiley Periodicals LLC.)

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel-Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel-Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

Chronic disease or injury of the vasculature impairs the functionality of vascular wall cells particularly in their ability to migrate and repair vascular surfaces. Under pathologic conditions, vascular endothelial cells (ECs) lose their non-thrombogenic properties and decrease their motility. Alternatively, vascular smooth muscle cells (SMCs) may increase motility and proliferation, leading to blood vessel luminal invasion. Current therapies to prevent subsequent blood vessel occlusion commonly mechanically injure vascular cells leading to endothelial denudation and smooth muscle cell luminal migration. Due to this dichotomous migratory behavior, a need exists for modulating vascular cell growth and migration in a more targeted manner. Here, we examine the efficacy of utilizing small direct current electric fields to influence vascular cell-specific migration ("galvanotaxis"). We designed, fabricated, and implemented an in vitro chamber for tracking vascular cell migration direction, distance, and displacement under galvanotactic influence of varying magnitude. Our results indicate that vascular ECs and SMCs have differing responses to galvanotaxis; ECs exhibit a positive correlation of anodal migration while SMCs exhibit minimal change in directional migration in relation to the electric field direction. SMCs exhibit less motility response (i.e. distance traveled in 4 h) compared to ECs, but SMCs show a significantly higher motility at low electric potentials (80 mV/cm). With further investigation and translation, galvanotaxis may be an effective solution for modulation of vascular cell-specific migration, leading to enhanced endothelialization, with coordinate reduced smooth muscle in-migration., (Copyright © 2020 Elsevier Inc. All rights reserved.)