Cytotoxicity,oxidative stress and inflammation induced by ZnO nanoparticles in endothelial cells: interaction with palmitate or lipopolysaccharide

Recent studies showed that ZnO nanoparticles (NPs) might induce the toxicity to human endothelial cells. However, little is known about the interaction between ZnO NPs and circulatory components, which is likely to occur when NPs enter the blood. In this study, we evaluated ZnO NP‐induced cytotoxicity, oxidative stress and inflammation in human umbilical vein endothelial cells (HUVECs), with the emphasis on the interaction with palmitate (PA) or lipopolysaccharide (LPS), because PA and LPS are normal components in human blood that increase in metabolic diseases. Overall, ZnO NPs induced cytotoxicity and intracellular reactive oxygen species (ROS) at a concentration of 32 μg ml⁻¹, but did not significantly affect the release of inflammatory cytokines or adhesion of THP‐1 monocytes to HUVECs. In addition, exposure to ZnO NPs dose‐dependently promoted intracellular Zn ions in HUVECs. PA and LPS have different effects. Two hundred μm PA significantly induced cytotoxicity and THP‐1 monocyte adhesion, but did not affect ROS or release of inflammatory cytokines. In contrast, 1 μg ml⁻¹ LPS significantly induced ROS, release of inflammatory cytokines and THP‐1 monocyte adhesion, but not cytotoxicity. The presence of ZnO NPs did not significantly affect the toxicity induced by PA or LPS. In addition, the accumulation of Zn ions after ZnO NP exposure was not significantly affected by the presence of PA or LPS. We concluded that there was no interaction between ZnO NPs and PA or LPS on toxicity to HUVECs in vitro . Copyright © 2016 John Wiley & Sons, Ltd.     

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO₂ NPs were used to compare the effects. Exposure to 32?μg/mL ZnO NPs (p?2 NPs (p?>?0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO₄ induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO₂ NP exposure (p?>?0.05). The presence of 250?nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p?p?>?0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p?>?0.05) except neutral red uptake assay (p?相似文献   

The size‐dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO₂‐NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis‐block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO₂‐NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2‐related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 μg/mL), all the four sizes of TiO₂‐NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose‐dependent and negative size‐dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO₂‐NP‐treated groups. Intriguingly, a very similar manner of dose‐dependent and size‐dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO₂‐NP‐exposed HUVECs, with an inversely size‐dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO₂‐NP exposure would cause detrimental effects to human cardiovascular health.     

TiO2 nanoparticles induce endothelial cell activation in a pneumocyte–endothelial co-culture model

The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO₂ nanoparticles (TiO₂-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10 μg/cm² of TiO₂-NPs or 10 ng/mL of TNFα for 24 h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO₂-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8 h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24 h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO₂-NPs causes endothelial cell activation.     

Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation

Metal oxide nanoparticles are widely used in industry, cosmetics, and biomedicine. However, the effects of exposure to these nanoparticles on the cardiovascular system remain unknown. The present study investigated the effects of nanosized TiO₂ and ZnO particles on the migration and adhesion of monocytes, which are essential processes in atherosclerogenesis, using an in vitro set-up of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1). We also examined the effects of exposure to nanosized metal oxide particles on macrophage cholesterol uptake and foam cell formation. The 16-hour exposure to ZnO particles increased the level of monocyte chemotactic protein-1 (MCP-1) and induced the migration of THP-1 monocyte mediated by increased MCP-1. Exposure to ZnO particles also induced adhesion of THP-1 cells to HUVECs. Moreover, exposure to ZnO particles, but not TiO₂ particles, upregulated the expression of membrane scavenger receptors of modified LDL and increased cholesterol uptake in THP-1 monocytes/macrophages. In the present study, we found that exposure to ZnO particles increased macrophage cholesterol uptake, which was mediated by an upregulation of membrane scavenger receptors of modified LDL. These results suggest that nanosized ZnO particles could potentially enhance atherosclerogenesis and accelerate foam cell formation.     

Endothelial cell activation,oxidative stress and inflammation induced by a panel of metal-based nanomaterials

Abstract

The importance of composition, size, crystal structure, charge and coating of metal-based nanomaterials (NMs) were evaluated in human umbilical vein endothelial cells (HUVECs) and/or THP-1 monocytic cells. Biomarkers of oxidative stress and inflammation were assessed because they are important in the development of cardiovascular diseases. The NMs used were five TiO₂ NMs with different charge, size and crystal structure, coated and uncoated ZnO NMs and Ag which were tested in a wide concentration range. There were major differences between the types of NMs; exposure to ZnO and Ag resulted in cytotoxicity and increased gene expression levels of HMOX1 and IL8. The intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) expression were highest in TiO₂ NM-exposed cells. There was increased adhesion of THP-1 monocytic cells onto HUVECs with Ag exposure. None of the NMs increased the intracellular ROS production. There were no major effects of the coating of ZnO NMs. The TiO₂ NMs data on ICAM-1 and VCAM-1 expression suggested that the anatase form was more potent than the rutile form. In addition, the larger TiO₂ NM was more potent than the smaller for gene expression and ICAM-1 and VCAM-1 expression. The toxicological profile of cardiovascular disease-relevant biomarkers depended on composition, size and crystal structure of TiO₂ NMs, whereas the charge on TiO₂ NMs and the coating of ZnO NMs were not associated with differences in toxicological profile.     

Cytotoxicity,oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles

Abstract

Nanoparticles in primary form and nanoproducts might elicit different toxicological responses. We compared paint-related nanoparticles with respect to effects on endothelial oxidative stress, cytotoxicity and cell adhesion molecule expression. Primary human umbilical vein endothelial cells were exposed to primary nanoparticles (fine, photocatalytic or nanosized TiO₂, aluminium silicate, carbon black, nano-silicasol or axilate) and dust from sanding reference- or nanoparticle-containing paints. Most of the samples increased cell surface expressions of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), but paint sanding dust samples generally generated less response than primary particles of TiO₂ and carbon black. We found no relationship between the expression of adhesion molecules, cytotoxicity and production of reactive oxygen species. In conclusion, sanding dust from nanoparticle-containing paint did not generate more oxidative stress or expression of cell adhesion molecules than sanding dust from paint without nanoparticles, whereas the primary particles had the largest effect on mass basis.     

Protective effects of ligustrazine on TNF-α-induced endothelial dysfunction

To investigate the effects of Ligustrazine, a compound derived from chuanxiong, on tumor necrosis factor-α (TNF-α) stimulated endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α in vitro. Nitric oxide (NO) was measured as a standard of endothelial dysfunction. Two important indicators of autoimmunity, intracellular adhesion molecular-1 (ICAM-1) and heat shock protein 60 (HSP60), were selected to evaluate the influence of Ligustrazine on HUVECs. Ligustrazine (40 μg/ml) significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in HUVECs. The expressions of ICAM-1 and HSP60 were increased by TNF-α treatment, but dramatically inhibited by treatment with ligustrazine in TNF-α-stimulated cells. Ligustrazine increased the production of NO in HUVECs and had an immunomodulatory effect on HUVECs stimulated with TNF-α by down-regulating the expression of ICAM-1 and HSP60. These results suggest that ligustrazine protects the endothelium via inhibition of immunological reactions, preventing atherosclerosis.     

Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells.

Nebivolol, a third generation selective beta(1)-adrenoceptor (beta(1)-AR) antagonist, has been reported to reduce intracellular oxidative stress and to induce the release of nitric oxide (NO) from the endothelium. Nebivolol is also subjected to a complex metabolic process where glucuronidation, aromatic and alicyclic hydroxylation are the major pathways leading to several metabolites. We have studied the effect of nebivolol, its enantiomers and metabolites on intracellular oxidative stress and NO availability in human umbilical vein endothelial cells (HUVECs). Furthermore, since the receptors involved in this endothelial effect of nebivolol remain controversial, we have studied this matter by the use of antagonists of beta-AR. dl-Nebivolol, d-nebivolol and l-nebivolol significantly reduced the formation of reactive oxygen species (ROS) and superoxide induced by oxidized-low density lipoprotein (ox-LDL), although the racemic and l-form were significantly more active than d-nebivolol in this activity. A marked decrease in the availability of intracellular NO was found in HUVECs exposed to ox-LDL and this parameter was normalized by the prior incubation with dl-nebivolol, d-nebivolol and l-nebivolol; the effect of racemate was mainly mimicked by its l-enantiomer. eNOS activity significantly increased by a 5-min contact of HUVECs with dl-nebivolol and l-nebivolol, but not with d-nebivolol, and a similar pattern was observed when the intracellular calcium increase was measured. The metabolites A2, A3', A12 and A14 but not A1, A3 and R 81,928, derived from different metabolic pathways, retained the antioxidant activity of the parent racemic compound dl-nebivolol, reducing the intracellular formation of ROS and superoxide. The effects of dl-nebivolol on intracellular formation of NO, eNOS activity and intracellular Ca(2+) were partially antagonized by the antagonists of beta(1-2)-AR nadolol or by the beta(3)-AR antagonist SR59230A and further antagonized by their combination or by (beta(1-2-3)-AR antagonist bupranolol. In conclusion, this study shows that the NO releasing effect of nebivolol is mainly due to its l-enantiomer; the racemate and its enantiomers possess a remarkable antioxidant activity that contributes to its effect on the cellular NO metabolism and the activation of beta(3)-AR through a calcium dependent pathway is involved in the mechanisms leading to the NO release.     

Motorcycle exhaust particles up-regulate expression of vascular adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells

Epidemiological studies have shown that there is a strong correlation between atherosclerosis and ambient air pollution. In this study, we found that motorcycle exhaust particles (MEP) induced adhesion between cells of the human monocytic leukemia cell line (THP-1) and human umbilical vein endothelial cells (HUVECs) in a time-and dose-dependent manner. In addition, MEP treatment induced both mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HUVECs. The IκB degradation and p65 nuclear translocation was found in MEP-treated HUVECs, suggested the involvement of nuclear factor-κB (NF-κB). MEP-induced VCAM-1 and ICAM-1 protein expression was inhibited by NF-κB inhibitor BAY 11-7085. Oxidative stress was also involved in the signaling of VCAM-1 and ICAM-1 expression. MEP treatment caused hydrogen peroxide and superoxide formation. Pretreatment with α-tocopherol could inhibit MEP-induced reactive oxygen intermediates generation and suppressed MEP-induced IκB degradation and adhesion molecules expression. Furthermore, the carbon black (CB) nanoparticles with different diameters could induce VCAM-1 and ICAM-1 protein expression; however, polycyclic aromatic hydrocarbons (PAHs) only increased the expression of ICAM-1 but not that of VCAM-1 in HUVECs. In this study, we found that MEPs could induce ICAM-1 and VCAM-1 expression through oxidative stress and NF-κB activation in HUVECs.     

Angiostatin Inhibition of Vascular Endothelial Growth Factor– Stimulated Nitric Oxide Production in Endothelial Cells

Angiostatin (AS), a proteolytic fragment of plasminogen, is a potent antiangiogenic factor. It was reported that AS attenuates the vasodilatory response to vascular endothelial growth factor (VEGF) in isolated interventricular arterioles. Here, we investigated the effect of AS on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). AS inhibited VEGF-stimulated NO production in a dose-dependent manner, whereas AS alone did not affect basal NO production. Disruption of kringle structures by reduction of disulfide bonds resulted in the loss of the inhibitory effect of AS on VEGF-stimulated NO production. To elucidate how AS might impair VEGF activation of endothelial NO synthase (eNOS), we further examined whether AS would affect Ca²⁺-dependent and -independent pathways of eNOS activation. AS had no effect on the transient increase in cytosolic Ca²⁺ levels elicited by VEGF. In contrast, AS prevented VEGF-potentiated eNOS phosphorylation at Ser1177. These results clearly indicate that AS inhibits VEGF-stimulated NO production in HUVECs without affecting basal NO production. The kringle structures of AS are required for this effect, and impairment of Ser1177 phosphorylation of eNOS might be involved in the inhibition of VEGF-stimulated NO production by AS.     

Expression of adhesion molecules, monocyte interactions and oxidative stress in human endothelial cells exposed to wood smoke and diesel exhaust particulate matter

Toxicological effects of wood smoke particles are less investigated than traffic-related combustion particles. We investigated the effect of wood smoke particles, generated by smouldering combustion conditions, on human umbilical endothelial cells (HUVECs) co-cultured with or without monocytic THP-1 cells. Standard reference material (SRM) 2975 diesel exhaust particles were used as benchmark particles. Wood smoke particles at 50 μg/ml or 100 μg/ml caused adhesion of THP-1 monocytes onto HUVECs in co-cultures, whereas SRM2975 had no such effect. Both types of particles from 1 μg/ml increased VCAM-1 expression on HUVECs in mono-cultures. However, only the exposure to wood smoke particles was associated with increased expression of TNF and IL8 mRNA in THP-1 cells. We found no effect on the intracellular production of reactive oxygen species by the fluorescent probe DCFH-DA, whereas especially the wood smoke particles caused increased level of DNA strand breaks and oxidised guanines at concentrations with low cytotoxicity. In conclusion, our results indicate that the adherence of monocytes on endothelial cells in wood smoke particle exposed cultures depend on activation of both cell types.     

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.     

Cryptotanshinone attenuates in vitro oxLDL-induced pre-lesional atherosclerotic events

Development of early stage atherosclerosis involves the activation of endothelial cells by oxidized low-density lipoprotein (oxLDL) with subsequent increases in endothelial permeability and expression of adhesion molecules favoring the adherence of monocytes to the endothelium. Cryptotanshinone (CTS), a major compound derived from the Chinese herb Salvia miltiorrhiza, is known for its protective effects against cardiovascular diseases. The aim of this study was to determine whether CTS could prevent the oxLDL-induced early atherosclerotic events. OxLDL (100?μg/mL) was used to increase endothelial permeability and induce monocyte-endothelial cell adhesion in human umbilical vein endothelial cells (HUVECs). Endothelial nitric oxide (NO) concentrations, a permeability-regulating molecule, and expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured. Results show that a) endothelial hyperpermeability was suppressed by 94?% (p?相似文献   

Beneficial effects of evodiamine on P2X4-mediated inflammatory injury of human umbilical vein endothelial cells due to high glucose

Evodiamine has been reported to exhibit anti-inflammatory and anti-nociceptive effects, but the underlying mechanisms remain to be defined. P2X₄ receptor (P2X₄R) is a subtype of ATP receptors and plays important roles in pain, inflammatory and immune responses. We aimed to investigate whether evodiamine has beneficial effects on endothelial inflammatory injury mediated by chronic high glucose condition. We found that culturing human umbilical vein endothelial cells (HUVECs) with high glucose significantly increased the expression of P2X₄ receptor in HUVECs, cytosolic Ca^2 + concentrations and intracellular reactive oxygen species (ROS) while decreasing nitric oxide (NO); these effects could be reversed by evodiamine. High glucose also significantly increased the expression of the pro-inflammatory activators (NF-κB) and TNFR-ɑ, which was accompanied by the elevation of P2X₄R levels. Evodiamine was able to down-regulate the elevated NF-κB, TNFR-ɑ, P2X₄R and ROS, and up-regulate the decreased NO_. Thus the evodiamine may exert the anti-inflammation activity on high-glucose challenge HUVEC via suppressing the P2X4R signaling pathway, exhibiting beneficial ability to protect HUVECs from glucotoxicity.     

Protective effects of eugenol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells.

Eugenol, a natural constituent of a number of aromatic plants and their essential oil fractions, has several biological effects. However, its protective effects against endothelial injury remain unclarified. This study investigates how eugenol affects human umbilical vein endothelial cells (HUVECs) dysfunction mediated by oxidized low density lipoprotein (oxLDL). Our results showed that the suppression of endothelial NO synthase (eNOS) expression, enhancement of adhesion molecules (ICAM, VCAM, and E-selectin) expression, and adherence of monocytic THP1 cells caused by a non-cytotoxic concentration (100 microg/ml) of oxLDL were ameliorated following a eugenol treatment (12.5-100 microM) in HUVECs. Eugneol also inhibited the reactive oxygen species (ROS) generation, intracellular calcium accumulation, and the subsequent mitochondrial membrane potential collapse, cytochrome c release and caspase-3 activation induced by oxLDL. The cytotoxicity and apoptotic features induced by a cytotoxic concentration (200 microg/ml) of oxLDL was also attenuated by eugenol. Our results suggest that eugenol may protect against the oxLDL-induced dysfunction in endothelial cells.     

Sargachromenol protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation

Vascular inflammation is a key factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the protective effects of sargachromenol (SCM) against tumor necrosis factor (TNF)-α-induced vascular inflammation. SCM decreased the expression of cell adhesion molecules, including intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs), resulted in reduced adhesion of monocytes to HUVECs. SCM also decreased the production of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 in TNF-α-induced HUVECs. Additionally, SCM inhibited activation of nuclear factor kappa B (NF-κB) induced by TNF-α through preventing the degradation of inhibitor kappa B. Moreover, SCM reduced the production of reactive oxygen species in TNF-α-treated HUVECs. Overall, SCM alleviated vascular inflammation through the regulation of NF-κB activation and through its intrinsic antioxidant activity in TNF-α-induced HUVECs. These results indicate that SCM may have potential application as a therapeutic agent against vascular inflammation.     

Inhibitory effects of sepiapterin on vascular endothelial growth factor-A-induced proliferation and adhesion in human umbilical vein endothelial cells

Tetrahydrobiopterin (BH₄) has been known to be an essential cofactor for the activities of nitric oxide (NO) synthase and aromatic amino acid hydroxylases, which are involved in physiological and pathological processes. In the present study, we report that sepiapterin, the more stable form of BH4 precursor, modulates vascular endothelial growth factor-A (VEGF-A)-induced cell proliferation and adhesion in human umbilical vein endothelial cells (HUVECs). The antiproliferative activity of sepiapterin in VEGF-A-treated HUVECs is associated with inhibition of the expression of cyclin-dependent kinases (Cdks) such as Cdk4 and Cdk2. Pretreatment with NO synthase inhibitor does not abrogate the ability of sepiapterin to inhibit VEGF-A-induced cell proliferation and adhesion, indicating that the suppressive effects of sepiapterin on VEGF-Ainduced responses are mediated by NO-independent mechanism. Finally, we show that sepiapterin modulates VEGF-A-induced cell proliferation and adhesion through down-regulation of VEGF receptor-2 downstream signaling pathways. Taken together, these findings represent a novel function of sepiapterin in the regulation of angiogenesis, supporting further development and evaluation of sepiapterin as an antiangiogenic agent.