Anti-atherogenic mechanism of ethanol extract of Christia vespertilionis (L.f.) Bakh. F. Leaves in vitro.

BACKGROUND: Vascular inflammation is the key event in early atherogenesis. Pro-inflammatory endothelial cells induce monocyte recruitment into the sub-endothelial layer of the artery. This requires endothelial expression of adhesion molecules namely intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), alongside chemokines production. Christia vespertilionis (L.f.) Bakh.f. (CV) possesses anti-inflammatory property. However, its potential anti-atherogenic effect in the context of vascular inflammation has yet to be explored. PURPOSE: To evaluate the anti-atherogenic mechanism of 80% ethanol extract of CV leaves on tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs). METHODS: Qualitative analysis of the CV extract was carried out by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cell viability of HUVECs treated with CV extract was determined by MTT assay. The effect of CV extract on monocyte adhesion was determined by monocyte-endothelial adhesion assay. Protein expressions of ICAM-1, VCAM-1 and nuclear factor-kappa B (NF-κB) signaling pathway were determined by western blot while production of monocyte chemoattractant protein-1 (MCP-1) was determined by ELISA. RESULTS: LC-MS/MS analysis showed that CV extract composed of five main compounds, including schaftoside, orientin, isovitexin, 6-caffeoyl-D-glucose, and 3,3'-di-O-methyl ellagic acid. Treatment of CV extract at a concentration range from 5 to 60 µg/mL for 24 h maintained HUVECs viability above 90 %, therefore concentrations of 20, 40 and 60 µg/mL were selected for the subsequent experiments. All concentrations of CV extract showed a significant inhibitory effect on monocyte adhesion to TNF-α-activated HUVECs (p < 0.05). In addition, the protein expressions of ICAM-1 and VCAM-1 were significantly attenuated by CV in a concentration dependent manner (p < 0.001). At all tested concentrations, CV extract also exhibited significant inhibition on the production of MCP-1 (p < 0.05). Moreover, CV extract significantly inhibited TNF-α-induced phosphorylation of inhibitor of nuclear factor-κB kinase alpha/beta (IKKα/ß), inhibitor kappa B-alpha (IκBα), NF-κB and nuclear translocation of NF-κB (p < 0.05). CONCLUSION: CV extract inhibited monocyte adhesion to endothelial cells by suppressing protein expressions of cell adhesion molecules and production of chemokines through downregulation of NF-κB signaling pathway. Thus, CV has the potential to be developed as an anti-atherogenic agent for early treatment of atherosclerosis.

Anti-Atherosclerotic Properties of Aronia melanocarpa Extracts Influenced by Their Chemical Composition Associated with the Ripening Stage of the Berries.

The high content of bioactive compounds in Aronia melanocarpa fruit offers health benefits. In this study, the anti-atherosclerotic effect of Aronia extracts was assessed. The impact on the level of adhesion molecules and the inflammatory response of human umbilical vein endothelial cells (HUVECs) was shown in relation to the chemical composition and the stage of ripening of the fruits. Samples were collected between May (green, unripe) and October (red, overripe) on two farms in Poland, which differed in climate. The content of chlorogenic acids, anthocyanins, and carbohydrates in the extracts was determined using HPLC-DAD/RI. The surface expression of ICAM-1 and VCAM-1 in HUVECs was determined by flow cytometry. The mRNA levels of VCAM-1, ICAM-1, IL-6, and MCP-1 were assessed using the quantitative real-time PCR method. The farms' geographical location was associated with the quantity of active compounds in berries and their anti-atherosclerotic properties. Confirmed activity for green fruits was linked to their high chlorogenic acid content.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

Actinidia arguta Extract Containing Myo-Inositol Suppresses TNF-α-Induced VCAM-1 Expression and Monocyte Adhesion to Endothelial Cells via Inhibition of the PTEN/Akt/GSK-3ß and NF-κB Signaling Pathways.

The primary inflammatory process in atherosclerosis, a major contributor to cardiovascular disease, begins with monocyte adhering to vascular endothelial cells. Actinidia arguta (kiwiberry) is an edible fruit that contains various bioactive components. While A. arguta extract (AAE) has been recognized for its anti-inflammatory characteristics, its specific inhibitory effect on early atherogenic events has not been clarified. We used tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs) for an in vitro model. AAE effectively hindered the attachment of THP-1 monocytes and reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) in HUVECs. Transcriptome analysis revealed that AAE treatment upregulated phosphatase and tensin homolog (PTEN), subsequently inhibiting phosphorylation of AKT and glycogen synthase kinase 3ß (GSK3ß) in HUVECs. AAE further hindered phosphorylation of AKT downstream of the nuclear factor kappa B (NF-κB) signaling pathway, leading to suppression of target gene expression. Oral administration of AAE suppressed TNF-α-stimulated VCAM-1 expression, monocyte-derived macrophage infiltration, and proinflammatory cytokine expression in C57BL/6 mouse aortas. Myo-inositol, identified as the major compound in AAE, played a key role in suppressing THP-1 monocyte adhesion in HUVECs. These findings suggest that AAE could serve as a nutraceutical for preventing atherosclerosis by inhibiting its initial pathogenesis.

Celecoxib attenuates interleukin 33-induced expression of vascular cell adhesion molecule-1 in human ovarian endometriotic stromal cells.

OBJECTIVE: Endometriosis is an estrogen-dependent chronic inflammatory disease in women of reproductive age. A review of the literature revealed that cytokines and inflammatory factors are associated with endometriosis-associated infertility. Interleukin 33 (IL-33) is a strong inducer of other pro-inflammatory cytokines. Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in recruiting inflammatory cells, whose expression facilitates leukocyte adhesion and is rapidly induced by pro-inflammatory cytokines. Many studies have indicated that VCAM-1 expression is high in endometriosis; however, whether the expression of VCAM-1 is related to IL-33 is unclear. MATERIALS AND METHODS: Human ovarian endometriotic stromal cells (hOVEN-SCs) were treated with IL-33 to enable investigation of cell characterization, gene and protein expression, and signal pathways. Proliferation potential was measured using an MTT assay. Gene expression was analyzed using reverse transcription-polymerase chain reaction. Protein expression assay was performed using western blot analysis. RESULTS: This study investigated the effects of IL-33 on VCAM-1 and COX-2 expression in hOVEN-SCs. First, the results revealed that the IL-33/ST2/mitogen-activated protein kinase (MAPK) signaling pathway could increase the expression of VCAM-1 and COX-2 in hOVEN-SCs. Second, we discovered that COX-2 expression was essential for IL-33-induced VCAM-1 expression because the effects could be negated through NS398, a selective COX-2 inhibitor. Finally, treatment of IL-33-treated hOVEN-SCs with celecoxib significantly and dose-responsively decreased VCAM-1 expression. CONCLUSION: Taken together, these results indicate that IL-33 can upregulate VCAM-1 expression in hOVEN-SCs through the IL-33/ST2/MAPK/COX-2 signaling pathway and thereby contribute to endometriosis.

Saponins from Allium macrostemon Bulbs Attenuate Endothelial Inflammation and Acute Lung Injury via the NF-κB/VCAM-1 Pathway.

Endothelial inflammation is a multifaceted physiological process that plays a pivotal role in the pathogenesis and progression of diverse diseases, encompassing but not limited to acute lung infections like COVID-19, coronary artery disease, stroke, sepsis, metabolic syndrome, certain malignancies, and even psychiatric disorders such as depression. This inflammatory response is characterized by augmented expression of adhesion molecules and secretion of pro-inflammatory cytokines. In this study, we discovered that saponins from Allium macrostemon bulbs (SAMB) effectively inhibited inflammation in human umbilical vein endothelial cells induced by the exogenous inflammatory mediator lipopolysaccharide or the endogenous inflammatory mediator tumor necrosis factor-α, as evidenced by a significant reduction in the expression of pro-inflammatory factors and vascular cell adhesion molecule-1 (VCAM-1) with decreased monocyte adhesion. By employing the NF-κB inhibitor BAY-117082, we demonstrated that the inhibitory effect of SAMB on VCAM-1 expression may be attributed to the NF-κB pathway's inactivation, as characterized by the suppressed IκBα degradation and NF-κB p65 phosphorylation. Subsequently, we employed a murine model of lipopolysaccharide-induced septic acute lung injury to substantiate the potential of SAMB in ameliorating endothelial inflammation and acute lung injury in vivo. These findings provide novel insight into potential preventive and therapeutic strategies for the clinical management of diseases associated with endothelial inflammation.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

Circulating levels of miR125a, miR126, and miR146a-5p in patients with obstructive sleep apnea and their relation with markers of endothelial dysfunction.

BACKGROUND: obstructive sleep apnea (OSA) is a prevalent sleep disorder that is associated with increased risk factors for cardiovascular diseases (CVDs). Oxidative stress, insulin resistance, inflammation, and endothelial dysfunction are increased in OSA patients and microRNAs (miRs) are regulatory elements that influence these pathological mechanisms. miR125a, miR126, and miR146a-5p play a role in these pathological mechanisms and have not been evaluated in patients with OSA. METHOD: This case-control study was performed on 90 OSA patients and 34 controls. Circulating levels of miR125a, miR126, and miR146a-5 were determined using real-time PCR, and serum levels of hsCRP, ICAM-1, and VCAM-1 were evaluated using ELISA kits. RESULTS: miR125a and miR146a were elevated in patients with OSA compared to controls while miR126 decreased significantly. All three miRs indicated a remarkable difference between the mild-OSA group compared to the severe-OSA group. Furthermore, patients with OSA showed elevated levels of hsCRP, ICAM-1, and VCAM-1. Multiple linear regression indicated an independent association of miR125a with ICAM-1 and hsCRP, miR126 associated with VCAM-1 and total cholesterol, and miR146a-5p represented an association with apnea-hypopnea index and ICAM-1. Furthermore, miR146a-5p illustrated a good diagnostic ability to differentiate between OSA and controls. CONCLUSIONS: Circulating miR125a, miR126, and miR146a-5p fluctuations in patients with OSA and their relations with markers of endothelial dysfunction provide in vivo evidence and suggest a potential role for these miRs with endothelial dysfunction in patients with OSA.

Aberrant cytokine and VCAM-1 expression in patients with viral and non-viral related liver cirrhosis.

The study aim was to compare the alterations in the expression levels of proinflammatory and chemotactic cytokines as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-17A and IL-8, the down regulatory cytokine IL-10, in addition to the vascular cell adhesion molecule-1 (VCAM-1) gene in different groups of patients with cirrhosis due to various etiologies. This case-control study included 84 patients suffering from cirrhosis of viral and non-viral etiologies and 20 sex and age-matched healthy controls. All patients were subjected to detailed history taking, clinical examination, and liver function assessment. The expression levels of TNF-α, IL-17A, IL-8, IL-10, and VCAM-1 were assessed in peripheral blood mononuclear cells by real-time PCR. Patients with cirrhosis showed marked changes in the tested gene expression levels relative to the control group. Higher expression levels of all genes except IL-10 were seen in patients of the viral than in the non-viral groups. Most of the significant correlations of liver function parameters were observed with TNF-α in both the viral and non-viral groups, followed by IL-17A. Increased TNF-α and IL-17A presented potential risk factors for disease progression to cirrhosis of Child class C.

Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts.

AIMS: Endothelial dysfunction plays a pivotal role in atherosclerosis, but the detailed mechanism remains incomplete understood. Nogo-B is an endoplasmic reticulum (ER)-localized protein mediating ER-mitochondrial morphology. We previously showed endothelial Nogo-B as a key regulator of endothelial function in the setting of hypertension. Here, we aim to further assess the role of Nogo-B in coronary atherosclerosis in ApoE-/- mice with pressure overload. METHODS AND RESULTS: We generated double knockout (DKO) mouse models of systemically or endothelium-specifically excising Nogo-A/B gene on an ApoE-/- background. After 7 weeks of transverse aortic constriction (TAC) surgery, compared to ApoE-/- mice DKO mice were resistant to the development of coronary atherosclerotic lesions and plaque rapture. Sustained elevation of Nogo-B and adhesion molecules (VCAM-1/ICAM-1), early markers of atherosclerosis, was identified in heart tissues and endothelial cells (ECs) isolated from TAC ApoE-/- mice, changes that were significantly repressed by Nogo-B deficiency. In cultured human umbilical vein endothelial cells (HUVECs) exposure to inflammatory cytokines (TNF-α, IL-1ß), Nogo-B was upregulated and activated reactive oxide species (ROS)-p38-p65 signaling axis. Mitofusin 2 (Mfn2) is a key protein tethering ER to mitochondria in ECs, and we showed that Nogo-B expression positively correlated with Mfn2 protein level. And Nogo-B deletion in ECs or in ApoE-/- mice reduced Mfn2 protein content and increased ER-mitochondria distance, reduced ER-mitochondrial Ca2+ transport and mitochondrial ROS generation, and prevented VCAM-1/ICAM-1 upregulation and EC dysfunction, eventually restrained atherosclerotic lesions development. CONCLUSION: Our study revealed that Nogo-B is a critical modulator in promoting endothelial dysfunction and consequent pathogenesis of coronary atherosclerosis in pressure overloaded hearts of ApoE-/- mice. Nogo-B may hold the promise to be a common therapeutic target in the setting of hypertension.

Epigenetic Mechanisms in Vascular Inflammation: Modulation of Endothelial Adhesion Molecules and Endothelium-Leukocyte Adhesion.

The endothelium, an essential component of the vascular system, plays a critical role in the inflammatory response. Under pro-inflammatory stimuli, endothelial cells undergo activation and dysfunction, leading to the release of inflammatory mediators and upregulation of cell adhesion molecules. These changes facilitate the adhesion, rolling, and transmigration of leukocytes into the subendothelial space. Emerging evidence suggests that epigenetic mechanisms, including nucleic acid methylation, post-translational histone modifications, and non-coding RNA, contribute significantly to the regulation of vascular inflammation and expression of cell adhesion molecules. Understanding the epigenetic molecular signatures that govern these processes may provide new insights into the development of therapeutic strategies to combat vascular inflammation and associated diseases. This review aims to summarize the current knowledge on the epigenetic mechanisms involved in modulating the intricate processes underlying vascular inflammation, with a specific focus on the expression of endothelial adhesion molecules and endothelium-leukocyte adhesion.

[Effect of mild moxibustion at 45 ℃ with different durations at "Zusanli" （ST36） on the inflammatory factors in the abdominal aorta of hyperlipidemia rats].

OBJECTIVE: To investigate the effects of mild moxibustion at 45°C on the chronic inflammatory response of the abdominal aorta in rats with hyperlipidemia and the effects of different moxibustion durations. METHODS: Thirty-six SD rats were randomly divided into the following groups： blank control group (2 weeks), model group (2 weeks), moxibustion group (2 weeks), blank group (4 weeks), model group (4 weeks), and moxibustion group (4 weeks). A model of hyperlipidemia with chronic inflammation was established through high-fat diet feeding for 8 weeks. Rats in the moxibustion groups received mild moxibustion treatment at bilateral "Zusanli"(ST36) at 45 °C, 10 min every time, once a day, for consecutive 2 or 4 weeks. The morphology of the abdominal aorta in each group was observed by using HE staining. Contents of serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), oxidized low-density lipoprotein (ox-LDL), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), endothelin-1 (ET-1) and the contents of nitric oxide (NO), ox-LDL, and ET-1 in the abdominal aorta were measured by using ELISA. Protein and mRNA expressions of IL-6 and TNF-α in the abdominal aorta of rats in each group were detected by using Western blot and real-time fluorescence quantitative PCR respectively. The positive expression of IL-6 in the abdominal aorta of rats was detected by Immunofluorescence. RESULTS: Compared to the blank control group, rats in the model group had increased contents of LDL, TC, TG, ox-LDL, VCAM-1, ICAM-1, IL-6, TNF-α, and ET-1 in the serum, increased contents of ox-LDL and ET-1 in the abdominal aorta, increased protein and mRNA expressions of IL-6 and TNF-α in the abdominal aorta(P<0.01, P<0.05, P<0.001), with decreased HDL content in the serum, decreased NO content in the abdominal aorta (P<0.01, P<0.05), as well as dark pink abdominal aorta, rough textures in the adventitia, media, and intima, and rough endothelial layer. Compared to the model group(2 weeks), LDL, ICAM-1, ET-1 contents in the serum, ox-LDL content in the abdominal aorta were decreased(P<0.05), while serum IL-6 and TNF-α contents, and NO content in the abdominal aorta were significantly increased(P<0.01, P<0.05), with smoother vascular walls, and relatively clear nucleus and surrounding tissue structures of abdominal aorta in the moxibustion group(2 weeks). Compared to the model group(4 weeks), contents of LDL, TC, TG, VCAM-1, ICAM-1, IL-6, TNF-α, ox-LDL, and ET-1 in the serum, ox-LDL and ET-1 contents in abdominal aorta, protein and mRNA expressions of IL-6 and TNF-α in the abdominal aorta were significantly decreased(P<0.05, P<0.01), while HDL content in the serum and NO content in the abdominal aorta were significantly increased(P<0.05, P<0.01), with smoother vascular walls, and relatively clear nucleus and surrounding tissue structures of abdominal aorta in the moxibustion group(4 weeks). In addition, content of HDL in the serum were significantly increased(P<0.05), while TNF-α content in the serum, protein expression of IL-6 in the abdominal aorta were significantly decreased (P<0.001, P<0.05), with smoother vascular walls, and clearer nucleus and surrounding tissue structures of abdominal aorta in the moxibustion group(4 weeks), in comparison with the moxibustion group(2 weeks). CONCLUSION: Mild moxibustion of 45 °C at ST36 can improve vascular endothelial damage and inflammatory response induced by high-fat diet by regulating serum lipids, vascular tone, adhesion molecules, and inflammatory factors, of which the effect of moxibustion intervention for 4 weeks is more significant.

Inducible nitric oxide synthase activity mediates TNF-α-induced endothelial cell dysfunction.

Inducible nitric oxide synthase (iNOS) and vascular endothelial dysfunction have been implicated in the development and progression of atherosclerosis. This study aimed to elucidate the role of iNOS in vascular endothelial dysfunction. Ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined with multivariate data analysis was used to characterize the metabolic changes in human umbilical vein endothelial cells (HUVECs) in response to different treatment conditions. In addition, molecular biology techniques were employed to explain the molecular mechanisms underlying the role of iNOS in vascular endothelial dysfunction. Tumor necrosis factor-α (TNF-α) enhances the expression of iNOS, TXNIP, and the level of reactive oxygen species (ROS) facilitates the entry of nuclear factor-κB (NF-κB) into the nucleus and promotes injury in HUVECs. iNOS deficiency reversed the TNF-α-mediated pathological changes in HUVECs. Moreover, TNF-α increased the expression of tumor necrosis factor receptor-2 (TNFR-2) and the levels of p-IκBα and IL-6 proteins and CD31, ICAM-1, and VCAM-1 protein expression, which was significantly reduced in HUVECs with iNOS deficiency. In addition, treating HUVECs in the absence or presence of TNF-α or iNOS, respectively, enabled the identification of putative endogenous biomarkers associated with endothelial dysfunction. These biomarkers were involved in critical metabolic pathways, including glycosylphosphatidylinositol-anchor biosynthesis, amino acid metabolism, sphingolipid metabolism, and fatty acid metabolism. iNOS deficiency during vascular endothelial dysfunction may affect the expression of TNFR-2, vascular adhesion factors, and the level of ROS via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.NEW & NOTEWORTHY Inducible nitric oxide synthase (iNOS) deficiency during vascular endothelial dysfunction may affect the expression of tumor necrosis factor receptor-2 and vascular adhesion factors via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.

The Impact of Hydroxychloroquine on Primary Feto-Placental Endothelial Cells from Healthy and Early-Onset Preeclamptic Placentas.

Hydroxychloroquine (HCQ), an anti-malarial drug, is suggested as a promising candidate for the treatment of pregnancy-related disorders associated with endothelial activation, among which there is preeclampsia (PE). Arterial feto-placental endothelial cells (fpECAs) were isolated from control (CTR) and early-onset preeclamptic (EO-PE) placentas. The aim of this study was to test potential protective effects of HCQ in an in vitro model of endothelial activation as well as in cells isolated from EO-PE placentas. To mimic PE conditions, CTR fpECAs were exposed to a pro-inflammatory environment consisting of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1ß (furtherly referred as MIX) with or without varying concentrations of HCQ (1 µg/mL and 10 µg/mL). Their effect on wound healing and endothelial barrier integrity was analyzed. Variations in the expression of IL-8 and leukocyte adhesion molecules (LAM) on both mRNA and protein levels were determined between CTR and PE fpECAs in the presence or absence of HCQ. MIX decreased wound healing and stability of the endothelial barrier, but HCQ did not affect it. Significant differences between CTR and EO-PE fpECAs were observed in IL-8 mRNA, protein secretion, and vascular cell adhesion protein 1 (VCAM-1) mRNA expression levels. After challenging CTR fpECAs with MIX, upregulation of both mRNA and protein levels was observed in all molecules. Combined treatment of HCQ and MIX slightly lowered VCAM-1 total protein amount. In CTR fpECAs, treatment with low concentrations of HCQ alone (1 µg/mL) reduced basal levels of IL-8 and VCAM-1 mRNA and secretion of IL-8, while in EO-PE fpECAs, a higher (10µg/mL) HCQ concentration slightly reduced the gene expression of IL-8. Conclusion: These results provide additional support for the safety of HCQ, as it did not adversely affect endothelial functionality in control fpECAs at the tested concentration. Furthermore, the observed limited effects on IL-8 secretion in EO-PE fpECAs warrant further investigation, highlighting the need for clinical trials to assess the potential therapeutic effects of HCQ in preeclampsia. Conducting clinical trials would offer a more comprehensive understanding of HCQ's efficacy and safety, allowing us to explore its potential benefits and limitations in a real-world clinical setting.

Reduced Tie2 in Microvascular Endothelial Cells Is Associated with Organ-Specific Adhesion Molecule Expression in Murine Health and Endotoxemia.

Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior.

Human IL-32θA94V mutant attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 via binding to cell surface receptor integrin αVß3 and αVß6 in TNF-α-stimulated HUVECs.

Interleukin-32 (IL-32), first reported in 2005, and its isoforms have been the subject of numerous studies investigating their functions in virus infection, cancer, and inflammation. IL-32θ, one of the IL-32 isoforms, has been shown to modulate cancer development and inflammatory responses. A recent study identified an IL-32θ mutant with a cytosine to thymine replacement at position 281 in breast cancer tissues. It means that alanine was also replaced to valine at position 94 in amino acid sequence (A94V). In this study, we investigated the cell surface receptors of IL-32θA94V and evaluated their effect on human umbilical vein endothelial cells (HUVECs). Recombinant human IL-32θA94V was expressed, isolated, and purified using Ni-NTA and IL-32 mAb (KU32-52)-coupled agarose columns. We observed that IL-32θA94V could bind to the integrins αVß3 and αVß6, suggesting that integrins act as cell surface receptors for IL-32θA94V. IL-32θA94V significantly attenuated monocyte-endothelial adhesion by inhibiting the expression of Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated HUVECs. IL-32θA94V also reduced the TNF-α-induced phosphorylation of protein kinase B (AKT) and c-jun N-terminal kinases (JNK) by inhibiting phosphorylation of focal adhesion kinase (FAK). Additionally, IL-32θA94V regulated the nuclear translocation of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), which are involved in ICAM-1 and VCAM-1 expression. Monocyte-endothelial adhesion mediated by ICAM-1 and VCAM-1 is an important early step in atherosclerosis, which is a major cause of cardiovascular disease. Our findings suggest that IL-32θA94V binds to the cell surface receptors, integrins αVß3 and αVß6, and attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs. These results demonstrate that IL-32θA94V can act as an anti-inflammatory cytokine in a chronic inflammatory disease such as atherosclerosis.

In vitro study of Nigella sativa and thymoquinone activity on endothelial activation and monocyte adhesion.

INTRODUCTION: Thymoquinone (TQ) is one of the bioactive compounds in Nigella sativa (NS). Also known as black seeds/cumin, it has been postulated to possess anti-atherogenic properties. However, research on the effects of NS oil (NSO) and TQ on atherogenesis remain scarce. The aim of this study is to determine gene and protein expression of Intercellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), and Endothelial-eukocyte adhesion molecule (E-selectin) in Human Coronary Artery Endothelial Cells (HCAECs). METHODS: HCAECs were stimulated for 24 hours (h) with 200 µg/ml of Lipopolysaccharides (LPS) and different concentrations of NSO (55, 110, 220, 440 µg/ml) or TQ (4.5, 9.0, 18.0, 36.0 µm). The effects of NSO and TQ on gene and protein expressions were measured using multiplex gene assay and ELISA assay, respectively. Rose Bengal assay was used to analyse monocyte binding activity. RESULTS: NSO and TQ significantly reduced ICAM-1 and VCAM-1 gene and protein expressions. TQ showed significant reduction activity of the biomarkers in dose dependent manner. HCAECs pre-treated with NSO and TQ for 24 h significantly lowered monocytes adherence compared to non-treated HCAECs. CONCLUSIONS: NSO and TQ supplementation have anti-atherogenic properties and inhibit monocytes' adherence to HCAECs via down-regulation of ICAM-1 expression. NSO could potentially be incorporated in standard treatment regimens to prevent atherosclerosis and its related complications.

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization.

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.