Expression In Human Umbilical Vein Endothelial Cells Research Articles

Oxytocin-induced endothelial nitric oxide dependent vasorelaxation and ERK1/2-mediated vasoconstriction in the rat aorta.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and plays an important role in the regulation of mammalian birth and lactation. It has been shown that oxytocin has important cardiovascular protective effects. Here we investigated the effects of oxytocin on vascular reactivity and underlying the mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and in rat aorta ex vivo. Oxytocin increased phospho-eNOS (Ser 1177) and phospho-Akt (Ser 473) expression in HUVECs in vitro and the aorta of rat ex vivo. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited oxytocin-induced Akt and eNOS phosphorylation. In the rat aortic rings, oxytocin induced a biphasic vascular reactivity: oxytocin at low dose (10-9–10-8 M) initiated a vasorelaxation followed by a vasoconstriction at high dose (10-7 M). L-NAME (a nitric oxide synthase inhibitor), endothelium removal or wortmannin abolished oxytocin-induced vasorelaxation, and slightly enhanced oxytocin-induced vasoconstriction. Atosiban, an oxytocin/vasopressin 1a receptor inhibitor, totally blocked oxytocin-induced relaxation and vasoconstriction. PD98059 (ERK1/2 inhibitor) partially inhibited oxytocin-induced vasoconstriction. Oxytocin also increased aortic phospho-ERK1/2 expression, which was reduced by either atosiban or PD98059, suggesting that oxytocin-induced vasoconstriction was partially mediated by oxytocin/V1aR activation of ERK1/2. The present study demonstrates that oxytocin can activate different signaling pathways to cause vasorelaxation or vasoconstriction. Oxytocin stimulation of PI3K/eNOS-derived nitric oxide may participate in maintenance of cardiovascular homeostasis, and different vascular reactivities to low or high dose of oxytocin suggest that oxytocin may have different regulatory effects on vascular tone under physiological or pathophysiological conditions.

Transcription factor FOXP1 mediates vascular endothelial dysfunction in diabetic retinopathy.

Diabetic retinopathy (DR) is still the fastest growing cause of blindness in working aged adults, and its typical characteristics are endothelial cell dysfunction and pericytes loss. Transcription factor fork head box P1 (FOXP1) is a member of FOX family involved in diabetes progression and is expressed in endothelial cells. The purpose of this study was to investigate the role and mechanism of FOXP1 in DR. The vitreous of DR patients and non-DR patients were collected, and the expression of FOXP1 was detected by real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) cultured in high glucose simulated DR environment, and the expressions of FOXP1, vascular endothelial growth factor (VEGF), and pigment epithelium derived factor (PEDF) were detected by RT-qPCR and western blot (WB) after transfection of small interfering RNA (siRNA) to knock out FOXP1. At the same time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), 5-ethynyl-2'-deoxyuridine assay (EDU), flow cytometry, Transwell assay, and tube-forming experiment were performed to determine cell proliferation, migration, and tube-forming ability. We found that FOXP1 was highly expressed in the vitreous of DR patients and HUVECs under high glucose condition. After FOXP1 was decreased, the activation of VEGF expression and inhibition of PEDF expression in HUVECs induced by high glucose were reversed; meanwhile, cell proliferation, migration, and tube formation decreased, and apoptosis was promoted. Generally, FOXP1 is highly expressed in the vitreous of DR patients, and its silence prevented VEGF/PEDF signaling pathway stimulated by high glucose and also reduced the proliferation, migration, and tube formation of endothelial cell, thus improving vascular endothelial dysfunction caused by DR. The results indicate that FOXP1 may be a therapeutic target of DR.

Activation of AMPK/miR-181b Axis Alleviates Endothelial Dysfunction and Vascular Inflammation in Diabetic Mice.

Hyperglycemia in diabetes mellitus impairs endothelial function and disrupts microRNA (miRNA) profiles in vasculature, increasing the risk of diabetes-associated complications, including coronary artery disease, diabetic retinopathy, and diabetic nephropathy. miR-181b was previously reported to be an anti-inflammatory mediator in vasculature against atherosclerosis. The current study aimed to investigate whether miR-181b ameliorates diabetes-associated endothelial dysfunction, and to identify potential molecular mechanisms and upstream inducer of miR-181b. We found that miR-181b level was decreased in renal arteries of diabetic patients and in advanced glycation end products (AGEs)-treated renal arteries of non-diabetic patients. Transfection of miR-181b mimics improved endothelium-dependent vasodilation in aortas of high fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, accompanied by suppression of superoxide overproduction and vascular inflammation markers. AMPK activator-induced AMPK activation upregulated miR-181b level in human umbilical vein endothelial cells (HUVECs). Chronic exercise, potentially through increased blood flow, activated AMPK/miR-181b axis in aortas of diabetic mice. Exposure to laminar shear stress upregulated miR-181b expression in HUVECs. Overall, our findings highlight a critical role of AMPK/miR-181b axis and extend the benefits of chronic exercise in counteracting diabetes-associated endothelial dysfunction.

Oxidative Stress-Induced Growth Inhibitor (OSGIN1), a Target of X-Box-Binding Protein 1, Protects Palmitic Acid-Induced Vascular Lipotoxicity through Maintaining Autophagy.

Saturated free fatty acids (FFAs) strongly correlate with metabolic syndromes and are well-known risk factors for cardiovascular diseases (CVDs). The mechanism of palmitic acid (PA)-induced vascular lipotoxicity under endoplasmic reticulum (ER) stress is unknown. In the present paper, we investigate the roles of spliced form of X-box-binding protein 1 (XBP1s) target gene oxidative stress-induced growth inhibitor 1 (OSGIN1) in PA-induced vascular dysfunction. PA inhibited the tube formation assay of primary human umbilical vein endothelial cells (HUVECs). Simultaneously, PA treatment induced the XBP1s expression in HUVECs. Attenuate the induction of XBP1s by silencing the XBP1s retarded cell migration and diminished endothelial nitric oxide synthase (eNOS) expression. OSGIN1 is a target gene of XBP1s under PA treatment. The silencing of OSGIN1 inhibits cell migration by decreasing phospho-eNOS expression. PA activated autophagy in endothelial cells, inhibiting autophagy by 3-methyladenine (3-MA) decreased endothelial cell migration. Silencing XBP1s and OSGIN1 would reduce the induction of LC3 II; therefore, OSGIN1 could maintain autophagy to preserve endothelial cell migration. In conclusion, PA treatment induced ER stress and activated the inositol-requiring enzyme 1 alpha–spliced XBP1 (IRE1α–XBP1s) pathway. OSGIN1, a target gene of XBP1s, could protect endothelial cells from vascular lipotoxicity by regulating autophagy.

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function.

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin–Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor—alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties.

Acute Administration of Metformin Protects Against Neuronal Apoptosis Induced by Cerebral Ischemia-Reperfusion Injury via Regulation of the AMPK/CREB/BDNF Pathway.

Metformin is a first-line anti-diabetic agent with a powerful hypoglycemic effect. Several studies have reported that metformin can improve the prognosis of stroke patients and that this effect is independent of its hypoglycemic effect; however, the specific mechanism remains unclear. In this research, we explored the effect and specific mechanism of metformin in cerebral ischemia-reperfusion (I/R) injury by constructing a transient middle cerebral artery occlusion model in vivo and a glucose and oxygen deprivation/reoxygenation (OGD/R) model in vitro. The results of the in vivo experiments showed that acute treatment with low-dose metformin (10 mg/kg) ameliorated cerebral edema, reduced the cerebral infarction volume, improved the neurological deficit score, and ameliorated neuronal apoptosis in the ischemic penumbra. Moreover, metformin up-regulated the brain-derived neurotrophic factor (BDNF) expression and increased phosphorylation levels of AMP-activated protein kinase (AMPK) and cAMP-response element binding protein (CREB) in the ischemia penumbra. Nevertheless, the above-mentioned effects of metformin were reversed by Compound C. The results of the in vitro experiments showed that low metformin concentrations (20 μM) could reduce apoptosis of human umbilical vein endothelial cells (HUVECs) under OGD/R conditions and promote cell proliferation. Moreover, metformin could further promote BDNF expression and release in HUVECs under OGD/R conditions via the AMPK/CREB pathway. The Transwell chamber assay showed that HUVECs treated with metformin could reduce apoptosis of SH-SY5Y cells under OGD/R conditions and this effect could be partially reversed by transfection of BDNF siRNA in HUVECs. In summary, our results suggest that metformin upregulates the level of BDNF in the cerebral ischemic penumbra via the AMPK/CREB pathway, thereby playing a protective effect in cerebral I/R injury.

Cadmium exposure enhances VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Vascular endothelium is a target of cadmium (Cd), which is a global pollutant of the environment. However, the detailed effects and underlying mechanisms remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with 0.1, 1, 5, 10, 50 µM cadmium chloride (CdCl2) for 12 h. It was found that vascular endothelial (VE)-cadherin mRNA and protein expression was upregulated by Cd in HUVECs in a dose-dependent manner. Higher levels of VE-cadherin were detected at cell-to-cell junctions in HUVECs treated with 10 µM CdCl2 compared with normal condition. The phosphorylation level of myosin-binding subunit of myosin phosphatase, a downstream substrate of Rho-associated protein kinase (ROCK), was reduced by 10 µM CdCl2, suggesting that Cd inhibited the Rho/ROCK pathway. Activation of ROCK by narciclasine reversed the Cd-induced increase of VE-cadherin expression. By contrast, ROCK pathway inhibitor Y27632 increased VE-cadherin expression in HUVECs. Following inhibition of the ROCK pathway, Cd did not significantly alter the level of VE-cadherin. Taken together, the results suggested that Cd exposure enhanced VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration and Proliferation Through Orai1-Mediated Calcium Signaling.

Parathyroid hormone is the main endocrine regulator of extracellular calcium and phosphorus levels. Secondary hyperparathyroidism–induced endothelial dysfunction may be related to calcium homeostasis disorders. Here, we investigated the effects of parathyroid hormone on human umbilical vein endothelial cells (HUVECs) and characterized the involvement of store-operated Ca2+ entry (SOCE) and the nuclear factor of activated T cells (NFAT) signaling pathway. We used immunoblot experiments to find that parathyroid hormone significantly enhanced the expression of the Orai1 channel, a type of channel mediating SOCE, SOCE activity, and Orai1-mediated proliferation of HUVECs but did not increase Orai2 and Orai3. RNA-seq was utilized to identify 1,655 differentially expressed genes (823 upregulated and 832 downregulated) in parathyroid hormone–treated HUVECs as well as enhanced focal adhesion signaling and expression levels of two key genes, namely, COL1A1 and NFATC1. Increased protein and mRNA expression levels of COL1A1 and NFATC1 were confirmed by immunoblotting and quantitative RT-PCR, respectively. Cytosol and nuclei fractionation experiments and immunofluorescence methods were used to show that parathyroid hormone treatment increased NFATC1 nuclear translocation, which was inhibited by a calcineurin inhibitor (CsA), a selective calmodulin antagonist (W7), an Orai channel inhibitor (BTP2), or Orai1 small interfering RNA (siRNA) transfection. Parathyroid hormone also increased COL1A1 expression, cell migration, and proliferation of HUVECs. The PTH-induced increase in HUVEC migration and proliferation were inhibited by CsA, W7, BTP2, or COL1A1 siRNA transfection. These findings indicated that PTH increased Orai1 expression and Orai1-mediated SOCE, causing the nuclear translocation of NFATC1 to increase COL1A1 expression and COL1A1-mediated HUVEC migration and proliferation. These results suggest potential key therapeutic targets of Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway in parathyroid hormone–induced endothelial dysfunction and shed light on underlying mechanisms that may be altered to prevent or treat secondary hyperparathyroidism–associated cardiovascular disease.

Hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BackgroundSteroid‐induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model.MethodsRT‐PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex‐treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual‐luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR‐7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa‐miR‐7974.ResultsIn our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex‐treated HUVECs cells, and the Dex‐mediated HUVEC apoptosis was impaired in hsa_circ_0058122‐silenced cells and increased in hsa_circ_0058122‐overexpressing cells. hsa_circ_0058122 competitively binds to hsa‐miR‐7974, which in turn interacts with insulin‐like growth factor binding protein 5 (IGFBP5).Conclusionshsa_circ_0058122/miR‐7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR‐7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR‐7974/IGFBP5 axis contributed to the Dex‐mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

ETS proto-oncogene 1 modulates PTP1B expression to participate in high glucose-mediated endothelial inflammation.

Hyperglycemia-induced endothelial inflammation participates in the pathogenesis of cardiovascular complications in diabetics. Previous studies showed that protein tyrosine phosphatase 1B (PTP1B) and ETS proto-oncogene 1 (ets1) are involved in hyperglycemia-induced endothelial inflammation. In this study, we hypothesized that ets1 modulates PTP1B expression, thus playing a crucial role in hyperglycemia-induced vascular endothelial inflammation. Our results indicated that high glucose increases monocyte/endothelial adhesion, vascular cell adhesion molecule-1 (VCAM-1) expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose-mediated endothelial inflammation is reversed by PTP1B silencing. In addition, high glucose increases ets1 expression in HUVECs. silencing reverses high glucose-mediated endothelial inflammation. Furthermore, the effect of ets1 overexpression is similar to that of high glucose treatment, which is counteracted by si-PTP1B. The results from ChIP assays indicated that ets1 occupies the PTP1B promoter region. Ets1 overexpression enhances PTP1B promoter activity, which is disappeared after specific binding site mutation. experiments demonstrated that the expressions of VCAM-1, PTP1B, and ets1, as well as the phosphorylation of p65 are augmented in the aorta of diabetic rats. In conclusion, ets1 contributes to hyperglycemia-mediated endothelial inflammation via upregulation of PTP1B expression.

Paeoniflorin alleviates NG-nitro-L-arginine methyl ester (L-NAME)-induced gestational hypertension and upregulates silent information regulator 2 related enzyme 1 (SIRT1) to reduce H2O2-induced endothelial cell damage

ABSTRACT Pregnancy-induced hypertension (PIH) is a leading cause of maternal mortality. Paeoniflorin has been reported to alleviate hypertension, thus relieving the injury of target organ. This study aimed to investigate the role of paeoniflorin in PIH development by regulating SIRT1 in rats. The mean arterial pressure (MAP), urine protein and histopathological damage of placenta in gestational hypertension rats were, respectively, detected by noninvasive tail-artery pressure measuring instrument, BCA method and H&E staining. The viability of human umbilical vein endothelial cells (HUVECs) treated with paeoniflorin or/and H2O2 was observed by CCK-8 assay. SIRT1 protein expression in HUVECs treated with paeoniflorin or/and H2O2 was analyzed by Western blot. Tunel assay, wound healing assay and tube formation assay were used to detect the apoptosis, migration and tube formation of HUVECs administrated with paeoniflorin or/and H2O2 or/and EX527 (SIRT1 inhibitor). As a result, MAP, urine protein and histopathological damage of placenta were enhanced in PIH rats, which were then alleviated by paeoniflorin. Paeoniflorin decreased the levels of sFlt-1, PlGF and VEGF in serum and placental tissues of gestational hypertension rats as well as the inflammatory response and oxidative stress. In addition, paeoniflorin promoted the expressions of SIRT1 and NO/eNOS and inhibited the production of iNOS in gestational hypertension rats to improve vascular endothelial cell injury. However, SIRT1 inhibition could suppress the protective effects of paeoniflorin on endothelial dysfunction of H2O2-induced HUVECs. In conclusion, paeoniflorin could improve gestational hypertension development by upregulating SIRT1.

In vitro evaluation of freeze-drying chitosan-mineralized collagen/Mg-Ca alloy composites for osteogenesis.

Magnesium (Mg) alloy with good mechanical properties and biodegradability is considered as one of the ideal bone repair materials. However, the rapid corrosion of Mg-based metals can pose harm to the function of an implant in clinical applications. In this study, micro-arc oxidation coating was prepared on the surface of the Mg-Ca matrix, then the chitosan and mineralized collagen (nano-hydroxyapatite/collagen; nHAC) were immobilized on the surface of the MAO/Mg-Ca matrix to construct the CS-nHAC/Mg-Ca composites of different component proportions (the ratio of CS to nHAC is 2:1, 1:1, and 1:2, respectively). The corrosion resistance, osteogenic activity, and angiogenic ability were extensively investigated. The results indicated that the CS-nHAC reinforcement materials can improve the corrosion resistance of the Mg matrix significantly and promote the proliferation and adhesion of mouse embryo osteoblast precursor cells (MC3T3-E1) and human umbilical vein endothelial cells (HUVECs). In addition, the CS-nHAC/Mg-Ca composites can not only promote the alkaline phosphatase (ALP) activity and extracellular matrix mineralization of MC3T3-E1 cells but also enhance the migration motility and vascular endothelial growth factor (VEGF) expression of HUVECs. Meanwhile, the 2CS-1nHAC/Mg-Ca composite exhibited the optimum function characteristics compared with other samples. Therefore, considering the improvement of corrosion resistance and biocompatibility, the CS-nHAC/Mg-Ca composites are expected to be a promising orthopedic implant.

Overexpression of hsa_circ_0022742 suppressed hyperglycemia-induced endothelial dysfunction by targeting the miR-503-5p/FBXW7 axis.

Type I and II diabetes adversely affect the microvasculature of several organs, although the regulatory mechanisms remain unclear. Previous studies have found that differentially expressed circRNAs associated with hyperglycemia (HG) induce endothelial dysfunction. In the present study, high-throughput sequencing was employed to assess abnormal circRNA expression in human umbilical vein endothelial cells (HUVECs) after HG treatment. Then, bioinformatics analysis, luciferase reporting analysis, angiogenic differentiation analysis, flow cytometry, and qRT-PCR analysis were performed to investigate the underlying regulatory mechanism and targets. The results demonstrate that hsa_circ_0022742 expression in HUVECs was decreased by HG treatment and overexpression of hsa_circ_0022742 suppressed HG-induced endothelial dysfunction. Luciferase analysis showed that miR-503-5p and FBXW7 were downstream targets of hsa_circ_0022742. Both overexpression of FBXW7 and inhibition of miR-503-5p reversed the protective effect of hsa_circ_0022742 against HG-induced endothelial dysfunction, including apoptosis, abnormal vascular differentiation, and secretion of inflammatory factors, indicating that hsa_circ_0022742 enhanced FBXW7 expression by sponging miR-503-5p. Taken together, these findings demonstrate that overexpression of hsa_circ_0022742 suppressed HG-induced endothelial dysfunction by targeting the miR-503-5p/FBXW7 axis.

Overexpression of sFlt-1 represses ox-LDL-induced injury of HUVECs by activating autophagy via PI3K/AKT/mTOR pathway.

Soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein, is involved in the pathogenesis of atherosclerosis (AS), and the underlying mechanism is still unclear. Here, we attempted to investigate the mechanism of action of sFlt-1 in AS. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low density lipoprotein (ox-LDL) to induce cell injury. ox-LDL treatment increased LC3-II/LC3-I ratio, Beclin-1 expression and GFP-LC3 puncta in HUVECs, suggesting that ox-LDL may induce autophagic flux impairment in HUVECs. ox-LDL-treated HUVECs displayed a decrease of sFlt-1 levels. Moreover, ox-LDL treatment reduced cell proliferation and elevated apoptosis in HUVECs, which was abrogated by sFlt-1 overexpression. Up-regulation of sFlt-1 repressed the activity of PI3K/AKT/mTOR signaling pathway and enhanced autophagy in HUVECs following ox-LDL treatment. Additionally, sFlt-1 overexpression-mediated increase of autophagy in ox-LDL-treated HUVECs was abolished by 3-methyladenine (autophagy inhibitor). 3-methyladenine abrogated the impact of sFlt-1 overexpression on proliferation and apoptosis in ox-LDL-treated HUVECs. This work confirmed that overexpression of sFlt-1 activated autophagy by repressing PI3K/Akt/mTOR signaling pathway, and thus alleviated ox-LDL-induced injury of HUVECs. Therefore, this study suggests that sFlt-1 may be a potential target for AS treatment.

Vitamin D decreases cell death and inflammation in human umbilical vein endothelial cells and placental explants from pregnant women with preeclampsia cultured with TNF-α

ABSTRACT This study evaluated the impact of vitamin D on Human Umbilical Vein Endothelial Cells (HUVEC) and inflammation in placental explants from women with preeclampsia (PE). HUVEC and explants from 10 late-onset PE (LOPE), 10 early-onset (EOPE), and 10 normotensive (NT) pregnant women were cultured with/without tumor necrosis factor (TNF-α) and VD. Interleukin-1β (IL-1β), 18 (IL-18), TNF-α, and TNF-related apoptosis-inducing ligand (TRAIL) were detected by ELISA. High mobility group box 1 (HMGB1) was determined by qPCR/Western blotting, and cell death by flow cytometry. Statistical significance was accepted at p < .05. Compared to the NT group, the endogenous levels of IL-1β, TNF-α, and IL-18 were higher in the PE group. The stimulus with TNF-α increased cytokines in NT, TNF-α in EOPE/LOPE, IL-18 in LOPE, and all cytokines in HUVEC. TNF-α+VD treatment decreased cytokines in explant and HUVEC supernatants. TRAIL was higher in EOPE versus NT, while TNF-α increased this receptor in NT versus control. In HUVEC, TNF-α increased TRAIL versus control, and TNF-α+VD decreased levels compared to only TNF-α stimulus. Protein expression of HMGB1 was higher in explant cultures treated with TNF-α and decreased after TNF-α+VD treatment in all groups, and gene/protein expression in HUVEC. Gene expression was elevated in EOPE versus NT and LOPE, and TNF-α increased HMGB1 in NT versus control, while TNF-α+VD decreased mRNA levels in EOPE. TNF-α stimulus increased late apoptosis in HUVEC, while VD increased viability. These in vitro observations suggest that VD administration to women with preeclampsia may be beneficial in reducing placental inflammation and cell death.

The Association and Pathogenesis of SERPINA3 in Coronary Artery Disease.

Background: Serine proteinase inhibitor A3 (SERPINA3) has been discovered in the pathogenesis of many human diseases, but little is known about the role of SERPINA3 in coronary artery disease (CAD). Therefore, we aim to determine its relationship with CAD and its function in the pathogenesis of atherosclerosis.Methods: In total 86 patients with CAD and 64 patients with non-CAD were compared. The plasma SERPINA3 levels were measured using ELISA. Logistic regression analysis and receiver-operating characteristic (ROC) analysis were performed to illustrate the association between plasma SERPINA3 levels and CAD. In vitro, real-time PCR (RT-PCR) and immunofluorescence staining were used to determine the expression of SERPINA3 in atherosclerotic plaques and their component cells. Then rat aortic smooth muscle cells (RASMCs) were transfected with siRNA to knock down the expression of SERPINA3 and human umbilical vein endothelial cells (HUVECs) were stimulated by SERPINA3 protein. EdU assay and scratch assay were used for assessing the capability of proliferation and migration. The cell signaling pathway was evaluated by western blot and RT-PCR.Results: Patients with CAD [104.4(54.5–259.2) μg/mL] had higher levels of plasma SERPINA3 than non-CAD [65.3(47.5–137.3) μg/mL] (P = 0.004). After being fully adjusted, both log-transformed and tertiles of plasma SERPINA3 levels were significantly associated with CAD. While its diagnostic value was relatively low since the area under the ROC curve was 0.64 (95% CI: 0.55–0.73). Secreted SERPINA3 might increase the expression of inflammatory factors in HUVECs. Vascular smooth muscle cells had the highest SERPINA3 expression among the aorta compared to endothelial cells and inflammatory cells. The knockdown of SERPINA3 in RASMCs attenuated its proliferation and migration. The phosphorylated IκBα and its downstream pathway were inhibited when SERPINA3 was knocked down.Conclusions: Elevated plasma SERPINA3 levels were associated with CAD. SERPINA3 can increase inflammatory factors expression in HUVECs. It can regulate VSMCs proliferation, migration, and releasing of inflammatory factors through the NF-κB signaling pathway. Thus, SERPINA3 played a significant role in the pathogenesis of atherosclerosis.

Preeclampsia and Gestational Hypertension: Biochemical and Antioxidant Features in Vitro Might Help Understand Different Outcomes.

Gestational hypertension (GH) is characterized by increased blood pressure after the 20th gestational week; the presence of proteinuria and/or signs of end-organ damage indicate preeclampsia (PE). Heme oxygenase-1 (HO-1) is an antioxidant enzyme with an important role in maintaining endothelial function, and induction of HO-1 by certain molecules shows potential in attenuating the condition's effects over endothelial tissue. HO-1 production can also be stimulated by potassium iodide (KI). Therefore, we evaluated the effects of KI over HO-1 expression in human umbilical vein endothelial cells (HUVECs) incubated with plasma from women diagnosed with GH or PE. Human umbilical vein endothelial cells were incubated with a pool of plasma of healthy pregnant women (n = 12), pregnant women diagnosed with GH (n = 10) or preeclamptic women (n = 11) with or without the addition of KI for 24 hours to evaluate its effect on this enzyme expression. Analysis of variance was performed followed by Dunnet's test for multiple comparisons between groups only or between groups with addition of KI (p ≤ 0.05). KI solution (1,000 µM) reduced HO-1 in the gestational hypertension group (p = 0.0018) and cytotoxicity in the preeclamptic group (p = 0.0143); treatment with KI reduced plasma cytotoxicity but did not affect the preeclamptic group's HO-1 expression. Our findings suggest that KI alleviates oxidative stress leading to decreased HO-1 expression; plasma from preeclamptic women did not induce the enzyme's expression in HUVECs, and we hypothesize that this is possibly due to inhibitory post-transcriptional mechanisms in response to overexpression of this enzyme during early pregnancy.

Thymosin β4 regulates endothelial cell function via activating the AKT pathway

The vascular eendothelial cells are highly heterogeneous and associated with numerous diseases. Thymosin β4 (Tβ4) plays pleiotropic roles in endothelial cell differentiation, migration and angiogenesis. However, the underlying mechanisms played by Tβ4 in the regulation of endothelial cells have not yet been well investigated. In the present study, Tβ4 -GFP adenovirus, transfected into human umbilical vein endothelial cells (HUVECs), and cell morphology were analyzed by fluorescence microscopy. ELISA was used to determine the concentration of Tβ4 expression. Furthermore, the effects of Tβ4 overexpression on HUVECs proliferation, apoptosis and migration were investigated. Real-time quantitative PCR and western blot were conducted to examine mRNA and protein expression in HUVECs with Tβ4 overexpression. Moreover, the underlying molecular mechanism of Tβ4 in HUVECs function was tested through treatment with LY294002, a PI3K/AKT inhibitor. Overexpression of Tβ4 increased the cell ability of HUVECs, and up-regulated the expression of the proliferation markers PCNA and Cyclin D1. In addition, overexpression of Tβ4 reduced HUVECs apoptosis, both under normoxic and hypoxic conditions. Moreover, overexpression of Tβ4 increased the ability of HUVECs to migrate through the membrane and up-regulated levels of MMP-2 and MMP-9. The use of LY294002 decreased the p-AKT (Ser473) level, which was induced by Tβ4 overexpression. Importantly, LY294002 reduced Tβ4-induced HUVECs proliferation and migration. In conclusion, our results suggest that Tβ4 is a major regulator of HUVECs function by activating the AKT signaling pathway.

A peptide derived from chaperonin 60.1, IRL201104, inhibits LPS-induced acute lung inflammation.

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1β and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

The Anti-Colon Cancer Effects of Essential Oil of Curcuma phaeocaulis Through Tumour Vessel Normalisation.

BackgroundNormalising tumour vessels had become a significant research focus in tumour treatment research in recent years. Curcumae rhizoma (CR) is an essential plant in traditional Chinese medicine as it promotes blood circulation and removes blood stasis. Similarly, CR improves local blood circulation.PurposeWe explored the anti-colon cancer effects of essential oil from CR (OCR) by investigating its role in normalising tumour vessels. We also provided a basis for research and development into new anti-cancer drugs.MethodsWe used colon cancer as a research focus to investigate OCR. We established an in vitro co-culture model of colon cancer cells and human umbilical vein endothelial cells (HUVEC). We also established an in vivo subcutaneous implant colon cancer model in nude mice. These studies allowed us to evaluate the comprehensive effects of OCR in in vivo and in vitro colon cancer and its role in normalising tumour blood vessels.Results In vitro, we found that OCR inhibited Human colon cancer cells (HCT116) and HUVEC cell proliferation and inhibited vascular endothelial growth factor-a (VEGFa) mRNA and protein expression in HUVECs in a co-culture system. Our in vivo studies showed that OCR inhibited colon cancer tumour growth, reduced angiogenesis in tumours and increased vascular endothelial (VE)-cadherin and pericyte coverage in tumour vessels.ConclusionsOCR inhibited colon cancer growth both in in vivo and in vitro models, reduced angiogenesis in tumours, improved tumour vessel structures and normalised tumour vessels.