Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation

Aim:

To investigate the effects Astragalus polysaccharides (APS) on tumor necrosis factor (TNF)-α-induced inflammatory reactions in human umbilical vein endothelial cells (HUVECs) and to elucidate the underlying mechanisms.

Methods:

HUVECs were treated with TNF-α for 24 h. The amounts of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined with Western blotting. HUVEC viability and apoptosis were detected using cell viability assay and Hoechst staining, respectively. Reactive oxygen species (ROS) production was measured by DHE staining. Monocyte and HUVEC adhesion assay was used to detect endothelial cell adhesive function. NF-κB activation was detected with immunofluorescence.

Results:

TNF-α (1-80 ng/mL) caused dose- and time-dependent increases of ICAM-1 and VCAM-1 expression in HUVECs, accompanied by significant augmentation of IκB phosphorylation and NF-κB translocation into the nuclei. Pretreatment with APS (10 and 50 μg/mL) significantly attenuated TNFα-induced upregulation of ICAM-1 VCAM-1 and NF-κB translocation. Moreover, APS significantly reduced apoptosis, ROS generation and adhesion function damage in TNF-α-treated HUVECs.

Conclusion:

APS suppresses TNFα-induced adhesion molecule expression by blocking NF-κB signaling and inhibiting ROS generation in HUVECs. The results suggest that APS may be used to treat and prevent endothelial cell injury-related diseases.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

3,4-oxo-isopropylidene-shikimic acid inhibits adhesion of polymorphonuclear leukocyte to TNF-α-induced endothelial cells in vitro

AIM：To examine the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (HUVEC) and explore its mechanism. METHODS:Adhesion of PMN to HUVEC was measured by rose bengal staining assay. Cell-EL1SA and RT-PCR methods were used to examine the expression of adhesion molecules ICAM-1. Cell viability was detected with MTT assay.RESULTS: ISA (1-100μmol/L) effectively reduced PMN adhesion to TNF-α-induced HUVEC with the inhibitory rate from 17.2% to 53.5%, and exerted no effect on PMN adhesion to normal HUVEC. Adhesion molecule ICAM-1 surface protein and mRNA expression induced by TNF-α (400kU/L) were significantly inhibited by ISA. In addition, the cell viability of HUVEC was unchanged 48h after treatment with ISA. CONCLUSION: ISA inhibited TNF-α-stimulated PMN-HUVEC adhesion and expression of ICAM-1.     

3,4-oxo-isopropylidene-shikimic acid inhibits adhesion of polymorphonuclear leukocyte to TNF-α-induced endothelial cells in υitro

INTRODUCTION Leukocyte infiltration to endothelium plays amajor role in the inflammatory response related tothrombosis, arteriosclerosis, and reperfusion injury, etc.Leukocyte infiltration requires a chain of reactions be-tween endothelial cells (EC) and leukocytes that initiallyretards intravascular leukocyte flow and finally leads toleukocyte transmigration through the endothelialmonolayer. These events are mediated by sequentialinteraction of different endothelial adhesion molecu…     

Cryptotanshinone inhibits TNF-α-induced LOX-1 expression by suppressing reactive oxygen species (ROS) formation in endothelial cells

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH₂-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.     

7,8-didehydrocimigenol from Cimicifugae rhizoma inhibits TNF-α-induced VCAM-1 but not ICAM-1expression through upregulation of PPAR-γ in human endothelial cells

Activators of PPAR have been demonstrated to inhibit the induction of VCAM-1 but not ICAM-1 in human endothelial cells (EC). During the screening of anti-inflammatory activity of traditional herbs, we found 7,8-didehydrocimigenol (7,8-DHC), one of active triterpenoids of Cimicifugae rhizoma (C. rhizoma) increases PPAR-γ expression in EC in a time- and dose-dependent manner. Therefore, we asked whether 7,8-DHC selectively inhibits the expression of VCAM-1 but not ICAM-1 in TNF-α-activated EC via upregulation of PPAR-γ. Treatment with 7,8-DHC or PPAR-γ agonists (GW1929, troglitazone) inhibited the expression of VCAM-1 but not ICAM-1. Furthermore, the selective inhibition of VCAM-1 expression was inhibited by PPAR-γ antagonist, GW9662, or siPPAR-γ-transfected cells. 7,8-DHC significantly inhibited NF-kB activity via inhibition of phosphorylation of IkB and it also inhibited phosphorylation of ERK1/2 and Akt but not PKC. Finally, attachment of monocytes (U937) to EC by TNF-α was significantly reduced by 7,8-DHC. These results indicate that upregualtion of PPAR-γ by 7,8-DHC in EC inhibits NF-kB activity of TNF-α-activated EC which leads to selective inhibition of VCAM-1 expression. In addition, ERK1/2 and Akt signal pathways are involved in differential regulation by 7,8-DHC. We concluded that 7,8-DHC can be used for the treatment of cardiovascular disorders such as atherosclerosis.     

Bp5250 inhibits vascular endothelial growth factor-induced angiogenesis and HIF-1α expression on endothelial cells

Angiogenesis plays a critical role in many physiological and pathological phenomena. A number of anti-angiogenesis drugs have been used in the clinical treatment of diseases such as malignant tumors and macular degeneration. Vascular endothelial growth factor (VEGF), the major pro-angiogenesis factor, is known to stimulate various steps of endothelial angiogenic activity, such as proliferation, migration, and differentiation into vessel-like tubes. In this study, we tested the effects of bp5250 on the angiogenesis of human umbilical endothelial cells (HUVECs). Bp5250 suppressed VEGF-induced endothelial cell proliferation by triggering apoptosis, and reduced endothelial cell migration toward VEGF. Bp5250 also decreased VEGF-stimulated tube formation and rat aortic ring sprouting on Matrigel in a concentration-dependent manner. In the VEGF-activated signaling pathways, bp5250 decreased the phosphorylation of ERK, p38, PI3K-AKT, Src, and FAK and also reduced the activation of the cytoskeleton-associated Rho family, all in a concentration-dependent manner. Bp5250 also attenuated the hypoxia-inducible factor-1α (HIF-1α) and VEGF-stimulated mRNA expression of HUVECs under the hypoxic condition. In vivo, angiogenesis was restrained by a daily intraperitoneal administration of bp5250 in a dose-dependent manner (1–3 mg/kg/d) in the Matrigel plug implantation assay. These results indicate that bp5250 is a potential candidate for developing anti-angiogenic agents.     

Rimonabant inhibits TNF-α-induced endothelial IL-6 secretion via CB1 receptor and cAMP-dependent protein kinase pathway

Aim:

To investigate whether rimonabant, a cannabinoid receptor antagonist, had inhibitory effects on inflammatory reactions in human umbilical vein endothelial cells (HUVEC).

Methods:

TNF-α-induced IL-6 production was measured by ELISA and effects on related signaling pathways were investigated by immunoblot analysis. Cellular cAMP level was measured using kinase-coupled luciferase reaction.

Results:

Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IκB kinase (IKK) α/β and IκB-α degradation. ACEA, a cannabinoid receptor subtype 1 (CB1) agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of cAMP-dependent protein kinase (PKA), abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production.

Conclusion:

Rimonabant had anti-inflammatory effects on endothelial cells and inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect was related to CB1 antagonism and PKA activation.     

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy.     

Catechol, a bioactive degradation product of salicortin, reduces TNF-α induced ICAM-1 expression in human endothelial cells

The phenolic glucoside salicortin was isolated from a Willow bark extract, and its ability to reduce the TNF- α induced ICAM-1 expression (10 ng/mL, 30 min pretreatment with salicortin) was tested IN VITRO on human microvascular endothelial cells (HMEC-1). After 24 h, 25 μM salicortin decreased the TNF- α induced ICAM-1 expression to 65.9 % compared to cells which were treated only with TNF- α. In parallel, the stability of 25 μM salicortin under assay conditions was determined by HPLC. Within 24 h, the salicortin concentration decreased to 3.1 μM whereas catechol, a known NF- κB inhibitor, rose as a metabolite. After 8 h the catechol concentration was relatively constant and varied between 8.2 and 10.9 μM. Considering this degradation in the IN VITRO test system, 10 μM catechol was added 8 h after TNF- α stimulation, and 16 h later the ICAM-1 expression was determined. In this setting, the ICAM-1 expression was reduced to 74.8 %. This is comparable to the effect obtained from 25 μM salicortin and indicates that its activity is related to the generation of catechol, as salicin, saligenin, and salicylic acid are only marginally active or inactive in this test system in a concentration up to 50 μM. These results indicate catechol as an important bioactive metabolite from salicortin.     

Tetrandrine inhibits Ca^2＋-activated chloride channel in cultured human umbilical vein endothelial cells

INTRODUCTION Tetrandrine (Tet, 6,6',7,12-tetramethoxy-2,2'- dim-ethyl-berbaman) is a purified bis-benzylisoquinoline al-kaloid derivedfrom the root ofa Chinese herb (Stephaniatetrandra S Moore)[1,2]. It was first shown as an anti-hypertensive agent in both normal and hypertensivesubjects in 1950s[3,4]. The primary anti-hypertensiveaction of Tet is presumably due to its vasodilatoryproperty, which was confirmed both in vivo (15 mg/kg in conscious rats) and in vitro (1-100 μmol/L,effecti…     

Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release

Aim： Paeonol （2＇-hydroxy-4＇-methoxyacetophenone） from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells （VECs） in vitro.
Methods： VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 （miR-21） and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-α release by VECs was measured by ELISA.

Results： Ox-LDL treatment inhibited VEC growth in dose- and time-dependent manners （the value of IC50 was about 20 mg/L at 24 h）. Furthermore, ox-LDL （20 mg/L） significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL （20 mg/L） significantly increased the release of TNF-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol （120 μmol/L） prevented ox-LDL-induced increases in miR-21 expression and TNF-α release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN＇s 3＇-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment.

Conclusion： miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.     

Hyperoside inhibits lipopolysaccharide-induced inflammatory responses in microglial cells via p38 and NFκB pathways

Hyperoside (quercetin-3-O-β-d-galactoside) is an active compound isolated from herbs. Neuroinflammation is a key mechanism involved in neurodegenerative disorders including Parkinson's disease. In this study, we aimed to investigate the potentiality of hyperoside in inhibiting microglia-mediated neuroinflammation. BV2 microglial cells were pretreated with hyperoside and stimulated with lipopolysaccharide (LPS). The results showed that hyperoside significantly inhibited LPS-induced production of nitric oxide and pro-inflammatory cytokines including IL-1β and TNF-α, as well as the expression of inducible nitric oxide synthase. Similar results were observed in primary microglial cells isolated from neonatal mice. Analyses in MAPK and NFκB signaling combined with specific inhibitors suggested that hyperoside attenuated the LPS-induced inflammatory responses via p38 and NFκB pathways. Furthermore, hyperoside suppressed reactive microglia-mediated neurotoxicity as evidenced by conditioned media culture, but had no direct impact on MPP⁺-induced toxicity in SH-SY5Y neuroblastoma cells. Collectively, our data suggest that hyperoside may serve as a protective agent by alleviating microglia activation in disorders such as Parkinson's disease.     

Sertraline ameliorates inflammation in CUMS mice and inhibits TNF-α-induced inflammation in microglia cells

Evidence indicates that inflammation plays a crucial role in depression. Therefore, new antidepressants might be identified by screening drugs for their anti-inflammatory actions. Sertraline hydrochloride (SERT), a widely used antidepressant, has anti-inflammatory effects in clinical studies, but the mechanism involved is unclear. In this study, we used cell and molecular biology to determine the possible anti-inflammatory mechanism of SERT in vivo and in vitro. Experimental data from the in vivo study showed that mice exposed to chronic unpredictable mild stress (CUMS) had significantly higher levels of major inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β] and inducible nitric oxide synthase [iNOS]) in peripheral and central tissues compared with the control group. Treatment of CUMS mice with SERT significantly reduced the levels of these inflammatory cytokines and inhibited the phosphorylation of nuclear factor-κB (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). Moreover, SERT reduced serum levels of transaminase in CUMS mice. Our in vitro study revealed that SERT suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. SERT also inhibited the TNF-α-induced nuclear translocation of NF-κB by inhibiting IκB-α phosphorylation. Furthermore, SERT inhibited TNF-α-induced inflammatory cytokines in BV2 microglia cells. SERT directly bound to TNF-α and TNF-α receptor 1 (TNFR1) to potently block TNF-α/TNFR1-triggered signaling. These results indicate that SERT might treat depression by inhibiting the activation of microglia via the NF-κB signaling pathway. This study provides a basis for the research and development of antidepressants that act to reduce inflammation and the expression of inflammatory mediators.     

Telmisartan attenuates hyperglycemia-exacerbated VCAM-1 expression and monocytes adhesion in TNFα-stimulated endothelial cells by inhibiting IKKβ expression

Uncontrolled hyperglycemia accelerates endothelial damage and vascular inflammation caused by proinflammatory cytokines including tumor necrosis factor α (TNFα), which leads to arteriosclerotic cardiovascular diseases such as myocardial infarction. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is prescribed for treatment of hypertensive patients with concurrent diabetes mellitus (DM). Although a few clinical trials have suggested that telmisartan decreases cardiovascular complications in diabetic patients, the molecular mechanism for the beneficial effects remains elusive. Here, we investigated a molecular mechanism and effects of telmisartan on the expression of vascular cell adhesion molecule-1 (VCAM-1) and attachment of monocytes onto endothelial cells induced by TNFα in hyperglycemia-treated bovine aortic endothelial cells (BAEC). Telmisartan dose-dependently decreased hyperglycemia-aggravated IκB kinase β (IKKβ) expression and nuclear factor-κB (NF-κB) p65-Ser⁵³⁶ phosphorylation, which accompanied a decrease in VCAM-1 expression and THP-1 monocytes adhesion. Among ARBs, including losartan and fimasartan, only telmisartan showed the inhibitory effects on expression of VCAM-1 and IKKβ, and phosphorylation of NF-κB p65-Ser⁵³⁶. The telmisartan's beneficial effects were not changed by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, although GW9662 clearly inhibited rosiglitazone-induced CD36 expression. Finally, ectopic expression of wild type (WT)-IKKβ significantly restored telmisartan-attenuated VCAM-1 expression, NF-κB p65-Ser⁵³⁶ phosphorylation, and THP-1 monocytes adhesion. Taken together, our findings demonstrate that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by decreasing expression of IKKβ and VCAM-1 independently of PPARγ. Telmisartan may be useful for the treatment of DM-associated vascular inflammation and cardiovascular diseases.     

Tanshinone ⅡA inhibits endothelin-1 production in TNF-α-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

Aim： To investigate the effects of tanshinone ⅡA （Tan ⅡA） on the regulation of the production of endothelin （ET）- 1 （including large ET- 1）, mRNA levels of ET- 1, endothelin-converting enzyme- 1 （ECE- 1）, endothelin-A receptor （ETA） and endothelin-B receptor （ETB） induced by TNF-α in rat brain microvascular endothelial cells （BMVEC）. Methods： The ET-1 release （including large ET-1） into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE- 1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. Results： The induction of ET- 1 release by TNF-α from cultured BMVEC was dose-dependently reduced by Tan ⅡA, but large ET-1 levels progressively increased in response to Tan ⅡA; the mRNA expression of ET- 1 was unaffected. Tan ⅡA also caused a decrease in ETA receptor mRNA and ECE- 1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-α alone or a combination of TNF-α and Tan ⅡA. Conclusion： These findings suggest that Tan ⅡA may inhibit ET-1 production in TNF-α-induced BMVEC through the suppression of ECE-1 synthesis.