Expression In Human Umbilical Vein Endothelial Cells Research Articles

Macrophage-derived exosomal miRNA-141 triggers endothelial cell pyroptosis by targeting NLRP3 to accelerate sepsis progression.

Sepsis, critical condition marked by severe organ dysfunction from uncontrolled infection, involves the endothelium significantly. Macrophages, through paracrine actions, play a vital role in sepsis, but their mechanisms in sepsis pathogenesis remain elusive. Objective: We aimed to explore how macrophage-derived exosomes with low miR-141 expression promote pyroptosis in endothelial cells (ECs). Exosomes from THP-1 cell supernatant were isolated and characterized. The effects of miR-141 mimic/inhibitor on apoptosis, proliferation, and invasion of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed using flow cytometry, CCK-8, and transwell assays. Key pyroptosis-related proteins, including caspase-1, IL-18, IL-1β, NLR Family Pyrin Domain Containing 3 (NLRP3), ASC, and cleaved-GSDMD, were analyzed via Western blot. The interaction between miR-141 and NLRP3 was studied using RNAhybrid v2.2 and dual-Luciferase reporter assays. The mRNA and protein level of NLRP3 after exosomal miR-141 inhibitor treatment was detected by qPCR and Western blot, respectively. Exosomes were successfully isolated. miR-141 mimic reduced cell death and pyroptosis-related protein expression in HUVECs, while the inhibitor had opposite effects, increasing cell death, and enhancing pyroptosis protein expression. Additionally, macrophage-derived exosomal miR-141 inhibitor increased cell death and pyroptosis-related proteins in HUVECs. miR-141 inhibits NLRP3 transcription. Macrophages facilitate sepsis progression by secreting miR-141 decreased exosomes to activate NLRP3-mediated pyroptosis in ECs, which could be a potentially valuable target of sepsis therapy.

Regulation of MMP-2 by IL-8 in Vascular Endothelial Cells: Probable Mechanism for Endothelial Dysfunction in Women with Preeclampsia.

Endothelial dysfunction (ED) in preeclampsia (PE) results from the convergence of oxidative stress, inflammation, and alterations in extracellular matrix components, affecting vascular tone and permeability. The molecular network leading to ED includes IL-8 and MMP-2. In vitro, IL-8 regulates the concentration and activity of MMP-2 in the trophoblast; this interaction has not been studied in endothelial cells during PE. We isolated human umbilical vein endothelial cells (HUVECs) from women with healthy pregnancies (NP, n = 15) and PE (n = 15). We quantified the intracellular concentration of nitric oxide and reactive oxygen species with colorimetric assays, IL-8 with ELISA, and MMP-2 with zymography and using an ELISA-type system. An IL-8 inhibition assay was used to study the influence of this cytokine on MMP-2 concentration and activity. HUVECs from women with PE showed significantly higher oxidative stress than NP. IL-8 and MMP-2 were found to be significantly elevated in PE HUVECs compared to NP. Inhibition of IL-8 in HUVECs from women with PE significantly decreased the concentration of MMP-2. We demonstrate that IL-8 is involved in the mechanisms of MMP-2 expression in HUVECs from women with PE. Our findings provide new insights into the molecular mechanisms regulating the ED distinctive of PE.

Helicobacter pylori-induced fibroblast-derived Serpin E1 promotes gastric cancer growth and peritoneal dissemination through p38 MAPK/VEGFA-mediated angiogenesis

BackgroundFibroblasts, especially cancer-associated fibroblasts (CAFs), represent the predominant stromal cell population in the tumor microenvironment and have an important function in tumorigenesis by interacting with tumor cells. However, their interaction remains elusive in an inflammatory tumor microenvironment induced by Helicobacter pylori (H. pylori).MethodsThe expression of Serpin family E member 1 (Serpin E1) was measured in fibroblasts with or without H. pylori infection, and primary gastric cancer (GC) cells. Serpin E1 knockdown and overexpression fibroblasts were generated using Serpin E1 siRNA or lentivirus carrying Serpin E1. Co-culture models of fibroblasts and GC cells or human umbilical vein endothelial cells (HUVECs) were established with direct contact or the Transwell system. In vitro functional experiments and in vivo tumorigenesis assay were employed to study the malignant behaviors of GC cells interacting with fibroblasts. ELISA was used for quantifying the levels of Serpin E1 and VEGFA in the culture supernatant. The tube formation capacity of HUVECs was assessed using a tube formation assay. Recombinant human Serpin E1 (recSerpin E1), anti-Serpin E1 antibody, and a MAPK pathway inhibitor were utilized to treat HUVECs for elucidating the underlying molecular mechanisms.ResultsSerpin E1 was predominantly expressed in gastric CAFs. H. pylori infection significantly enhanced the expression and secretion of Serpin E1 by CAFs. Both fibroblast-derived Serpin E1 and recSerpin E1 enhanced the growth, invasion, and migration of GC cells, along with increased VEGFA expression and tube formation in HUVECs. Furthermore, the co-inoculation of GC cells and fibroblasts overexpressing Serpin E1 triggered the expression of Serpin E1 in cancer cells, which facilitated together xenograft tumor growth and peritoneal dissemination of GC cells in nude mice, with an increased expression of Ki67, Serpin E1, CD31 and/or VEGFA. These processes may be mediated by Serpin E1-induced migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs.ConclusionH. pylori infection induces Serpin E1 expression in fibroblasts, subsequently triggering its expression in GC cells through their interaction. Serpin E1 derived from these cells promotes the migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs, thereby facilitating GC growth and peritoneal metastasis. Targeting Serpin E1 signaling is a potential therapy strategy for H. pylori-induced GC.

LncRNA MIR4697HG Alleviates Endothelial Cell Injury and Atherosclerosis Progression in Mice via the FUS/ANXA5 Axis.

Atherosclerosis (AS) manifests with arterial intimal injury, lipid deposition and chronic inflammation, which is a key pathogenic cause of cardio-cerebrovascular disorders. LncRNA MIR4697HG was downregulated in human advanced atherosclerotic plaques. This study probed the precise biological functions and downstream regulatory mechanisms of MIR4697HG during AS progression. MIR4697HG levels in atherosclerotic plaque tissues and normal arterial intima were measured by RT-qPCR. An injury model of human umbilical vein endothelial cells (HUVECs) was induced through treating with oxidative low-density lipoprotein (ox-LDL). MIR4697HG overexpression plasmids (pcDNA-MIR4697HG) was transfected into ox-LDL-treated HUVECs, and then cell viability, apoptosis, reactive oxygen species (ROS) level, oxidative stress marker protein malondialdehyde (MDA) level and superoxide dismutase (SOD) activity, and adhesion molecule intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels in HUVECs were determined. Moreover, the binding between MIR4697HG and fused in sarcoma (FUS) was checked with RNA pull-down assay. The interaction between FUS and annexin A5 (ANXA5) was gauged with Co-immunoprecipitation. Then MIR4697HG/FUS/ANXA5 axis mediated HUVEC functions were accessed with rescue experiments. Additionally, an AS model was established via feeding a high-fat diet for ApoE-/- mice, and lentivirus MIR4697HG overexpression vector (Lv-MIR4697HG) was injected into AS mice followed by detection of atherosclerotic plaque area in mice. MIR4697HG was downregulated in atherosclerotic plaque tissues and HUVECs stimulated by ox-LDL. MIR4697HG overexpression attenuated ox-LDL-induced HUVEC viability inhibition, apoptosis, oxidative stress and adhesion molecule release. Moreover, MIR4697HG bound with FUS and facilitated FUS expression in HUVECs. FUS knockdown abrogated the functions of lncRNA MIR4697HG overexpression in ox-LDL induced HUVEC injury. Besides, FUS could bind with ANXA5. FUS overexpression inhibited ox-LDL induced HUVEC injury, while ANXA5 knockdown reversed these effects. Additionally, Lv-MIR4697HG reduced atherosclerotic plaque area in ApoE-/- mice. LncRNA MIR4697HG mitigated ox-LDL-induced apoptosis, oxidative stress and adhesion molecule release in HUVECs and alleviated AS progression in mice through the FUS/ANXA5 axis.

Modified Hongyu Decoction promotes wound healing by activating the VEGF/PI3K/Akt signaling pathway.

Wound healing is a considerable problem for clinicians. Ever greater attention has been paid to the role of Chinese herbal monomers and compounds on wound healing. This study aims to elucidate the wound healing mechanism of Modified Hongyu Decoction (MHD) in vivo and in vitro. MHD wound healing activity in vivo was evaluated using an excision rat model. H and E staining, Masson's staining and immunofluorescence of wound tissue on days 7 and 14 were performed to evaluate the efficacy of MHD on wound healing. Subsequently, human umbilical vein endothelial cells (HUVECs) were used to evaluate wound healing characteristics in vitro. Cell Counting Kit-8 (CCK-8) and scratch assays were conducted to assess the effects of MHD on the proliferation and migration of HUVECs. The involvement of the VEGF/PI3K/Akt signaling pathway was assessed by western blotting. The rats in the MHD group displayed more neovascularization and collagen fibers. Western blotting of wound tissue showed that VEGF, PI3K, p-Akt and p-eNOS expression were significantly increased (p<0.05) in the MHD group. Cell Counting Kit-8 and scratch assays demonstrated that MHD promoted HUVECs proliferation and migration. MHD treatment significantly increased VEGF, PI3K, p-Akt and p-eNOS expression in HUVECs (p<0.05), which was inhibited by LY294002. Both in vivo and in vitro data indicated that MHD promotes wound healing by regulating the VEGF/PI3K/Akt signaling pathway.

Endothelial glycocalyx sensitivity to chemical and mechanical sub-endothelial substrate properties.

Glycocalyx (GCX) is a carbohydrate-rich structure that coats the surface of endothelial cells (ECs) and lines the blood vessel lumen. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, can be transduced and sent to ECs through mechanosensors such as GCX. Adverse stiffness alters GCX-mediated mechanotransduction and leads to EC dysfunction and eventually atherosclerotic cardiovascular diseases. To understand GCX-regulated mechanotransduction events, an in vitro model emulating in vivo vessel conditions is needed. To this end, we investigated the impact of matrix chemical and mechanical properties on GCX expression via fabricating a tunable non-swelling matrix based on the collagen-derived polypeptide, gelatin. To study the effect of matrix composition, we conducted a comparative analysis of GCX expression using different concentrations (60-25,000μg/mL) of gelatin and gelatin methacrylate (GelMA) in comparison to fibronectin (60μg/mL), a standard coating material for GCX-related studies. Using immunocytochemistry analysis, we showed for the first time that different substrate compositions and concentrations altered the overall GCX expression on human umbilical vein ECs (HUVECs). Subsequently, GelMA hydrogels were fabricated with stiffnesses of 2.5 and 5kPa, representing healthy vessel tissues, and 10kPa, corresponding to diseased vessel tissues. Immunocytochemistry analysis showed that on hydrogels with different levels of stiffness, the GCX expression in HUVECs remained unchanged, while its major polysaccharide components exhibited dysregulation in distinct patterns. For example, there was a significant decrease in heparan sulfate expression on pathological substrates (10kPa), while sialic acid expression increased with increased matrix stiffness. This study suggests the specific mechanisms through which GCX may influence ECs in modulating barrier function, immune cell adhesion, and mechanotransduction function under distinct chemical and mechanical conditions of both healthy and diseased substrates.

FUS regulates the alternative splicing of cell proliferation genes related to atherosclerosis.

FUS plays a significant role as an RNA-binding protein in several cellular processes, including RNA splicing, DNA repair, and transcriptional regulation. However, the RNA-binding capacity of FUS in atherosclerosis is unclear. We aimed to study the functions of FUS in inflammatory regulation through the role of the splicing factor. We knocked down FUS with siRNA to further study the overall transcriptional level and select alternative splicing (AS) of FUS regulation in human umbilical vein endothelial cells (HUVECs) by RNA sequencing. The results suggested that the knockdown of FUS significantly affected gene expression in HUVECs. In addition, the knockdown of FUS resulted in 200 differentially expressed genes (DEGs) that were highly related to apoptotic process, signal transduction, multicellular organism development, cell adhesion and regulation of transcription, and DNA-templated pathways. Importantly, FUS extensively regulated 2870 AS events with a significant difference. Functional analysis of its modulated AS genes revealed they were highly enriched in cell cycle and cell population proliferation pathways. The qRT-PCR and RNA-seq data showed consistent results. Our findings suggested new knowledge of the mechanisms of FUS associated with atherosclerosis.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM2.5 exposure

Cooking oil fume-derived PM2.5 (COF-PM2.5) is a major source of indoor air contamination in China, which has been demonstrated to be a hazard factor of cardiovascular and cerebrovascular diseases. This study aimed to investigate the role of ROS-mediated PERK/ATF4 signaling activation in COF-PM2.5-inhibited extracorporeal tube formation in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 100 μg/mL COF-PM2.5 at different times, with or without 100 nM PERK activity inhibitor GSK2606414 (GSK) or 200 μM antioxidant N-acetylcysteine (NAC) pretreatment. Our results showed that COF-PM2.5 exposure can inhibit extracorporeal tube formation and down-regulate VEGFR2 expression in HUVECs. Furthermore, our data indicated that COF-PM2.5 exposure can activate the PERK/ATF4 signaling in HUVECs. Mechanistically, pretreatment with GSK interdicted PERK/ATF4 signaling, thereby reversing COF-PM2.5-downregulated VEGFR2 protein expression in HUVECs. Furthermore, NAC reversed VEGFR2 expression downregulated induced by COF-PM2.5 by inhibiting the upregulation of intracellular ROS levels and PERK/ATF4 signaling in HUVECs. As above, COF-PM2.5 exposure could induce ROS release from HUVECs, which in turn activate the endoplasmic reticulum PERK/ATF4 signaling and inhibit tube formation of HUVECs.

Inhibitory Effects of Simvastatin on IL-33-Induced MCP-1 via the Suppression of the JNK Pathway in Human Vascular Endothelial Cells.

An alarmin, interleukin (IL)-33 is a danger signal that causes inflammation, inducing chemotactic proteins such as monocyte chemoattractant protein (MCP)-1 in various cells. As statins have pleiotropic actions including anti-inflammatory properties, we investigated the effects of simvastatin on IL-33-induced MCP-1 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with IL-33 in the presence or absence of simvastatin. Gene expression and protein secretion of MCP-1, phosphorylation of mitogen-activated protein kinase (MAPK), nuclear translocation of phosphorylated c-Jun, and human monocyte migration were investigated. Immunocytochemical staining and Western immunoblot analysis revealed that IL-33 augmented MCP-1 protein expression in HUVECs. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that IL-33 significantly increased MCP-1 mRNA and protein secretion, which were suppressed by c-jun N-terminal kinase (JNK) inhibitor SP600125 and p38 MAPK inhibitor SB203580. Simvastatin inhibited IL-33-induced MCP-1 mRNA, protein secretion, phosphorylation of JNK and c-Jun. Additionally, the IL-33-induced nuclear translocation of phosphorylated c-Jun and THP-1 monocyte migration were also blocked by simvastatin. This study demonstrated that IL-33 induces MCP-1 expression via the JNK and p38 MAPK pathways in HUVECs, and that simvastatin inhibits MCP-1 production by selectively suppressing JNK. Simvastatin may inhibit the progression of IL-33-induced inflammation via suppressing JNK to prevent MCP-1 production.

Effects of aurantiamide on a rat model of renovascular arterial hypertension.

Asperglaucide (ASP) is an aurantiamide, an effective constituent of purslane (Portulaca oleracea L.), a safe to eat greenery. Effects of ASP on endothelial function, endothelial nitric oxide synthase (eNOS) expression, vascular fluidity, renal and vascular reactive oxygen, and nitrogen species (ROS/RNS) production was examined in the two-kidney one-clip (2K-1C) rat model of renovascular arterial hypertension. ASP toxicity, dose dependent eNOS gene expression and protein levels were also analyzed in human umbilical vein endothelial cells (HUVEC). The 2K-1C model of hypertension was created via surgery and mean blood pressure (MBP) was measured by tail-cuff method during four weeks of ASP treatment. Erythrocyte deformability was monitored by rotational ektacytometry, while vascular constrictor and dilator responses were determined in organ baths. eNOS gene expression and protein levels were assessed in thoracic aorta and HUVEC. MBP was significantly decreased in hypertensive rats treated with ASP. Endothelium dependent vascular dilator and constrictor responses were also considerably improved following ASP treatment. There was a notable increase in red blood cell deformability in hypertensive rats treated with ASP as compared to hypertensive rats alone. A significant increase was observed in eNOS gene expression and protein levels in both normotensive and hypertensive rats treated with ASP. Treatment of HUVEC with 3µM ASP notably increased eNOS mRNA and protein levels. In conclusion, ASP lowered blood pressure, improved endothelium-mediated relaxation, decreased renovascular ROS/RNS production in hypertensive rats. ASP also increased eNOS protein expression in aorta and HUVEC at nontoxic doses. ASP may have future potential as an anti-hypertensive agent.

Exosomes from hypoxia-conditioned apical papilla stem cells accelerate angiogenesis in vitro through Notch/JAG1/VEGF signaling

Dental pulp angiogenesis is a committed step in pulp regeneration therapy, and exosomes provide a new cell-free choice for tissue regeneration. This study revealed the underlying regulatory mechanism of exosomes from stem cells of the apical papilla (SCAPs) under hypoxic state on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Exosomes extracted from normoxia or hypoxia-pretreated SCAPs were co-cultured with HUVECs, and hypoxia pretreatment increased the release of exosomes and the internalization of exosomes by HUVECs. Compared to normoxic SCAPs-derived exosomes, exosomes from hypoxic SCAPs were found to promote cell proliferation and migration in HUVECs, as it was respectively determined by Cell Counting Kit-8, RT-qPCR and Transwell assay. Besides, hypoxia-educated SCAPs-exosomes especially enhanced the angiogenesis abilities of HUVECs in vitro, which were confirmed by tube formation assay and RT-qPCR detection of angiogenesis-related molecular markers. Interestingly, we found that the hypoxia inducible factor-1α (HIF-1α)/Notch1 signaling pathway was activated in hypoxic SCAPs, and protein jagged-1 (JAG1) was delivered by hypoxic SCAPs-derived exosomes to increase vascular endothelial growth factor (VEGF) production in HUVECs. Moreover, exogenous interference of JAG1 expression in HUVECs partially neutralized the activities of hypoxic SCAPs-exosomes in promoting cell proliferation, migration and tube formation of HUVECs. In summary, this study elucidates that exosomes from hypoxic SCAPs shows high potential to promote angiogenesis in vitro through the HIF-1α/JAG1/VEGF signaling cascade, which may provide a new perspective for the development of vascular reconstruction measures during dental regeneration engineering.

Angiogenesis, coagulation, and fibrinolytic markers in acute promyelocytic leukemia (NB4): An evaluation of melatonin effects.

Melatonin is a powerful biological agent that has been shown to inhibit angiogenesis and also exerts anti-inflammatory effects. It is well known that new blood vessel formation (angiogenesis) has become an urgent issue in leukemia as well as solid tumors. Acute promyelocytic leukemia (APL) is a form of liquid cancer that manifests increased angiogenesis in the bone marrow of patients. Despite high-rate curable treatment with all-trans-retinoic acid (ATRA) and recently arsenic-trioxide (ATO), early death because of hemorrhage, coagulopathy, and Disseminated intravascular coagulation (DIC) remains still a concerning issue in these patients. It is, therefore, urgent to seek treatment strategies with antiangiogenic capabilities that also diminish coagulopathy and hyperfibrinolysis in APL patients. In this study, a coculture system with human umbilical vein endothelial cells (HUVECs) and NB4 APL cells was used to investigate the direct effect of melatonin on angiogenesis and its possible action on tissue factor (TF) and tissue-type plasminogen activator-1 (TPA-1) expression. Our experiments revealed that HUVEC-induced angiogenesis by cocultured NB4 cells was suppressed when melatonin alone or in combination with ATRA was added to the incubation medium. Melatonin at concentrations of 1 mM inhibited tube formation of HUVECs and also decreased interleukin-6 secretion and VEGF mRNA expression in HUVEC and NB4 cells. Taken together, the results of this study demonstrate that melatonin inhibits accelerated angiogenesis of HUVECs and ameliorates the coagulation and fibrinolysis indices stimulated by coculturing with NB4 cells.

Identification and Screening of Potential ACE2 Activating Peptides from Soybean Protein Isolate Hydrolysate against Ang II-Induced Endothelial Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is a counterregulator against ACE by converting angiotensin II (Ang II) to Ang-(1-7), and its down-regulation leads to endothelial dysfunction in the vascular system. In the present study, we investigated the effects of soybean protein isolate hydrolysate (SPIH) on Ang II-induced endothelial dysfunction with its underlying mechanisms via ACE2 activation in human umbilical vein endothelial cells (HUVECs). We further screened potential ACE2 activating peptides by peptidomics analysis combined with bioinformatics tools. Results showed that SPIH remarkably attenuated Ang II-induced cell migration from 129 to 92%, decreased the ROS level from 2.22-fold to 1.45-fold, and increased NO concentration from 31.4 ± 0.7 to 43.7 ± 0.1 μM in HUVECs. However, these beneficial effects were reversed by ACE2 inhibitor MLN-4760 to a certain extent, indicating the modulation of ACE2. Further results revealed that SPIH (1 mg/mL) significantly increased the expression and activity of ACE2 and two novel ACE2 activating peptides with different mechanisms were explored from SPIH. IVPQ and IAVPT (50 μM) enhanced ACE2 activity, and only IVPQ (50 μM) increased ACE2 protein expression in HUVECs. These findings furthered our understanding of the antihypertensive mechanism of SPIH mediating the ACE2 activation on vascular endothelium.

Modulation of the activation of endothelial nitric oxide synthase and nitrosative stress biomarkers by aspirin triggered lipoxins: A possible mechanism of action of aspirin in the antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is characterized by the clinical manifestation of vascular thrombosis (VT) or pregnancy morbidity (PM) and antiphospholipid antibodies (aPL) that can modify the nitric oxide production. Low-dose aspirin is used in the prevention and treatment of diverse alterations of pregnancy. One of the mechanisms of action of aspirin is to induce the production of aspirin-triggered-lipoxins (ATL). The aim of this study was to evaluate the modulatory effect of ATL over the activation of endothelial nitric oxide synthase (eNOS) and nitrosative stress biomarkers induced by aPL. We used polyclonal IgG and sera from women with aPL and PM/VT or VT only, and from women with PM only and positive for non-criteria aPL (SN-OAPS). In these sera, biomarkers of nitrosative stress (nitrites and nitrotyrosine) were measured. The protein expression of nitrotyrosine and the phosphorylation of eNOS (at Ser1177) were estimated in human umbilical vein endothelial cells (HUVECs) stimulated with polyclonal IgG with or without ATL. Women with SN-OAPS showed increased circulating levels of nitrites and nitrotyrosine. Likewise, polyclonal IgG from either SN-OAPS or VT patients stimulated nitrotyrosine expression in HUVECs. ATL decreased the nitrotyrosine expression induced by polyclonal IgG from the SN-OAPS group. ATL also recovered the reduced eNOS phosphorylation at Ser1177 in HUVECs stimulated with polyclonal IgG from women with PM/VT or SN-OAPS. Increased nitrosative stress present in serum of women with SN-OAPS is associated with IgG-mediated impaired endothelial NO synthesis in endothelial cells. ATL prevent these cellular changes.

Tp47-Induced Monocyte-Derived Microvesicles Promote the Adherence of THP-1 Cells to Human Umbilical Vein Endothelial Cells via an ERK1/2-NF-κB Signaling Cascade.

The Treponema pallidum membrane protein Tp47 induces immunocyte adherence to vascular cells and contributes to vascular inflammation. However, it is unclear whether microvesicles are functional inflammatory mediators between vascular cells and immunocytes. Microvesicles that were isolated from Tp47-treated THP-1 cells using differential centrifugation were subjected to adherence assays to determine the adhesion-promoting effect on human umbilical vein endothelial cells (HUVECs). Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in Tp47-induced microvesicle (Tp47-microvesicle)-treated HUVECs were measured, and the related intracellular signaling pathways of Tp47-microvesicle-induced monocyte adhesion were investigated. Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs (P < 0.01) and upregulated ICAM-1 and VCAM-1 expression in HUVECs (P < 0.001). The adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Tp47-microvesicle treatment of HUVECs activated the extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB signaling pathways, whereas ERK1/2 and NF-κB inhibition suppressed the expression of ICAM-1 and VCAM-1 and significantly decreased the adhesion of THP-1 cells to HUVECs. IMPORTANCE Tp47-microvesicles promote the adhesion of THP-1 cells to HUVECs through the upregulation of ICAM-1 and VCAM-1 expression, which is mediated by the activation of the ERK1/2 and NF-κB pathways. These findings provide insight into the pathophysiology of syphilitic vascular inflammation.

Ethanolic Extract of Garcinia kola Stem Bark Inhibits LDL-Uptake and LPS-Induced Anxa-1 and ICAM-1 Expression in Human Umbilical Vein Endothelial Cells (HUVECS)

The endothelial cells’ dysfunction linked to the development of atherosclerosis plays an important role in the regulation of inflammatory responses. Previous studies demonstrated the antiatherogenic effects of Garcinia kola seed extracts based on their lipid lowering effects. Our recent studies showed the in vitro antioxidant and anti-inflammatory activities of the ethanolic extract of Garcinia kola stem barks (EE). For more insight on the antiatherogenic effect of EE, we investigated its activity on some key points of the atherosclerosis process. The cytotoxicity of EE as well as its effects on LDL-uptake, LPS-induced InterCellular Adhesion Molecule (ICAM-1) and Annexin-1 (Anxa-1) expression and LPS-induced DNA damage were evaluated in Human Umbilical Vein Endothelial Cells (HUVECs). EE significantly (p&lt;0.0001) increased the cell viability in both naive and LPS-treated HUVECs. At the concentrations of 50 and 100 µg/ml, EE significantly reduced LDL-uptake by endothelial cells stimulated with LPS. The immunohistochemistry results showed a significant (p&lt;0.01) decrease in the ICAM-1 expression at the EE concentration of 250 µg/ml. EE also showed a significant (p&lt;0.0001) concentration-dependent reduction of Annexin-1 expression in LPS-treated HUVECs. Besides, EE exhibited significant inhibition on LPS-induced genotoxicity marked by a decrease in tail DNA expression (p&lt;0.0001) and tail movement expression (p&lt;0.001) for the concentrations of 50 and 100 µg/ml. These findings showed that EE may mitigate the atherogenic process by reducing LDL-uptake and the expression of adhesion molecules. Thus, the EE of Garcinia kola turns out to be a potential candidate for the treatment of atherosclerosis with a lower risk of toxicity.&#x0D; Keywords: Garcinia kola, LDL-uptake, LPS-induced inflammation, comet assay.

HSP90-Dependent Upregulation of EZH2 Promotes Hypoxia/Reoxygenation-Induced Pyroptosis by Inhibiting miR-22 in Endothelial Cells.

Endothelial cell pyroptosis induced by hypoxia/reoxygenation (H/R) plays a key role in the pathogenesis of myocardial infarction (MI). However, the underlying mechanism is not clearly elucidated. Human umbilical vein endothelial cells (HUVECs) exposed to H/R acted as in vitro model to investigate the mechanism of H/R-induced endothelial cell pyroptosis. CCK-8 assays were performed to investigate the viability of HUVECs. Calcein-AM/PI staining was carried out to quantify the death of HUVECs. The expression level of miR-22 was measured by RT-qPCR. The protein expression levels of zeste 2 polycomb repressive complex 2 subunit (EZH2), NLRP3, cleaved caspase-1 (c-caspase-1), GSDMD-N and heat shock protein 90 (HSP90) were measured by Western blot. Levels of IL-1β and IL-18 in culture medium were detected by ELISA. The intracellular localization of EZH2 was detected by immunofluorescence staining. Chromatin immunoprecipitation (ChIP) assay was used to detect the enrichment of EZH2 and H3K27me3 in the miR-22 promoter region. The binding between miR-22 and NLRP3 in HUVECs was confirmed by the dual luciferase assay. Reciprocal coimmunoprecipitation was conducted to detect the direct interaction between HSP90 and EZH2. H/R increased EZH2 expression, and the EZH2 siRNA could inhibit H/R-induced pyroptosis in HUVECs. H/R reduced miR-22 expression, which was reversed by EZH2 siRNA. Silencing of miR-22 by its inhibitor reversed EZH2 siRNA-induced pyroptosis inhibition in H/R-exposed HUVECs. Upregulation of miR-22 by its mimic suppressed EZH2 overexpression-enhanced pyroptosis in H/R-exposed HUVECs. ChIP assay confirmed that EZH2 bound to the miR-22 promoter region and repressed miR-22 expression through H3K27me3. Furthermore, luciferase reporter assay indicated that NLRP3 was a direct target of miR- 22 in HUVECs. Finally, HSP90 siRNA inhibited H/R-induced EZH2 expression, miR-22 downregulation, and pyroptosis in HUVECs. H/R induces pyroptosis via the HSP90/EZH2/miR-22/NLRP3 signaling axis in endothelial cells.

Oxidative Stress Elevates eNOS Expression and VSMCs Proliferation of the Umbilical Vein of GDM Mothers

Aim: Gestational diabetes mellitus (GDM) is associated with an increased risk of fetal and maternal complications, such astype 2 DM (diabetes mellitus) and cardiovascular disease (CVD). This study aimed to predict the potential for future vascular complications in mothers with GDM by evaluating oxidative stress, endothelial NO synthase (eNOS) expression, and vascular smooth muscle cell (VSMC) proliferation in the umbilical vessels of mothers with GDM. Material and Methods: Subjects were divided into two groups: the normoglycemic control (NGC) group (n = 10) and the GDM group (n = 12). Expression of eNOS and production of reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) were determined. The mitochondrial mass of HUVECs was evaluated by spectrofluorometry. VSMC proliferation was ascertained in vitro with an EdU cell proliferation assay. Advanced glycation end products (AGEs) accumulation was measured by ELISA and assessed by immunohistochemical staining Results: VSMC proliferation, eNOS expression, and ROS production in HUVECs were significantly increased, and greater immunohistochemical staining to AGEs was observed in endothelium in GDM. Conclusion: Increased oxidative stress, which elevates eNOS expression and VSMC proliferation in the umbilical vessels of mothers with GDM, may be a sign that mothers have a high potential for developing diabetes or cardiovascular disease in the future.

Protective Effects of Cirsilineol against Lipopolysaccharide-Induced Inflammation; Insights into HO-1, COX-2, and iNOS Modulation.

In this study, the potential protective effects of cirsilineol (CSL), a natural compound found in Artemisia vestita, were examined on lipopolysaccharide (LPS)-induced inflammatory responses. CSL was found to have antioxidant, anticancer, and antibacterial properties, and was lethal to many cancer cells. We assessed the effects of CSL on heme oxygenase (HO)-1, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). We also examined the effects of CSL on the expression of iNOS, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the pulmonary histological status of LPS-injected mice. The results showed that CSL increased HO-1 production, inhibited luciferase-NF-κB interaction, and reduced COX-2/PGE2 and iNOS/NO levels, leading to a decrease in signal transducer and activator of transcription (STAT)-1 phosphorylation. CSL also enhanced the nuclear translocation of Nrf2, elevated the binding activity between Nrf2 and antioxidant response elements (AREs), and reduced IL-1β expression in LPS-treated HUVECs. We found that CSL's suppression of iNOS/NO synthesis was restored by inhibiting HO-1 through RNAi. In the animal model, CSL significantly decreased iNOS expression in the pulmonary biostructure, and TNF-α level in the bronchoalveolar lavage fluid. These findings indicate that CSL has anti-inflammatory properties by controlling iNOS through inhibition of both NF-κB expression and p-STAT-1. Therefore, CSL may have potential as a candidate for developing new clinical substances to treat pathological inflammation.