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.     

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.     

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.     

Nitric oxide-mediated endothelial dysfunction--is there need to treat?

Before it gets to the development of manifest atherosclerotic lesions, all known risk factors primarily induce functional alterations in the vascular wall, namely in endothelial cells. Being termed endothelial dysfunction or endothelial activation, this condition is characterized by an altered availability of nitric oxide (NO). Under physiological conditions, NO is of unequivocal importance for the regulation of vascular homeostasis. Endothelium-derived NO released abluminally increases soluble guanylat cyclase activity in smooth muscle cells, thereby inducing relaxation and consequently vasodilatation. Intraluminally, NO inhibits the expression of adhesion molecules both on endothielial cells and neutrophils, thus preventing the adherence of cellular elements to the vascular wall. Furthermore, NO has antithrombotic effcts by inhibiting platelet aggregation and directly infuencing the synthesis of different factors involved in the coagulation cascade. Finally, in the long term, NO has been shown to exert antiproliferative properties. NO is generated intracellularly from L-arginine via NO-synthase with the help of several cofactors, including tetrahydobiopterin. Interestingly, it has recently become evident that under certain conditions, when there is a lack of tetrahydrobiopterin, NO-synthase produces reactive oxygen species instead of NO. Reactive oxygen species counteract the effects of NO and also scavenge NO resulting in the formation of peroxynitrite (ONOO). Peroxynitrite has been shown to have deleterious effects with respect to vascular function. The aim of the current review is to elucidate recent progress regarding the pathophysiological understanding of endothelial dysfunction. Furthermore, the significance of this condition for the evaluation and prognosis of patients is discussed. Finally, current therapeutical strategies in the treatment and improvement of endothelial dysfunction are highlighted.     

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

Evaluation of PPARγ agonists on rodent endothelial cell proliferation

The PPARγ agonist troglitazone (TG) induced an increased incidence of hemangiosarcomas in mice but was not carcinogenic in rats. In contrast, pioglitazone (PIO) did not induce hemangiosarcomas or any other tumors in mice. We previously demonstrated that TG increased the proliferation of endothelial cells (ECs) in liver and adipose tissue in mice, and acted as a mitogenic stimulant and an inhibitor of apoptosis in vitro in mouse, but not human, ECs. In the present study, we investigated whether TG had any effect on the proliferation of ECs in rats. We also evaluated the in vivo and in vitro effects of PIO on ECs in mice. In rats, TG did not increase the Ki-67 labeling index (LI) of ECs in liver or adipose tissue at doses used in the two-year bioassay, and did not increase hepatocyte proliferation. PIO administered to mice did not increase the Ki-67 LI of hepatocytes or ECs in liver or white adipose tissue, but slightly increased the EC proliferation in brown adipose tissue. PIO was slightly mitogenic on cultured mouse ECs after 3 days of treatment but not after 6 days, and there was no inhibition of apoptosis, in contrast to what was seen with TG. The data support the conclusion that sustained EC proliferation in mice is necessary, for the induction of hemangiosarcomas by TG, and these short-term and long-term effects are not seen with TG in the rat or with PIO in mice, treatments that also are not related to the induction of hemangiosarcomas in two-year bioassays.     

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.     

TiO₂ nanoparticles induce dysfunction and activation of human endothelial cells

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles-endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO?) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO? of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm2. Besides, TiO? induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO? induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.     

Emodin-6-O-β-d-glucoside down-regulates endothelial protein C receptor shedding

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway and in the cytoprotective pathway. Previously, EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). Soluble EPCR levels are increased in patients with systemic inflammatory diseases. Recently, we reported that a new active compound, emodin-6-O-β-d-glucoside (EG) from Reynoutria japonica, has anti-inflammatory activities. However, little is known of the effects of EG on EPCR shedding. Here, we investigated this issue by monitoring the effects of EG on the phorbol-12-myristate 13-acetate (PMA) or the cecal ligation and puncture (CLP)-mediated EPCR shedding and its underlying mechanisms. Data showed that EG potently inhibited the PMA and CLP-induced EPCR shedding by suppressing TACE expression. Given these results, EG could be used as a candidate therapeutic for the treatment of vascular inflammatory diseases.     

Oxidized low-density lipoproteins induce apoptosis in aortic and endocardial endothelial cells

目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导血管和心内膜内皮细胞凋亡．方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ·Ｌ－１氧化．观察ｏｘＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用．琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解．结果：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂量依赖性．环己米特和硫酸葡聚糖对此作用无影响．ＢＨＴ２０μｍｏｌ·Ｌ－１可取消ＤＮＡ降解．溶血性磷脂酰胆碱５０μｍｏｌ·Ｌ－１无诱导凋亡作用．ｏｘＬＤＬ诱导的ＤＮＡ降解可被依他酸取消．结论：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞凋亡．     

Interferon-γ-activated macrophages enhance angiogenesis from endothelial cells of rat aorta

The influence of interferon-γ (IFN-γ), an activator of macrophages, was investigated on angiogenesis in vitro from rat aortic endothelial cells (EC). Subcultured EC were cultured in 0.15% type I collagen gel with 2% fetal bovine serum (FBS)-Dulbecco's modified Eagle's medium. Tube formation by EC began on the first day of culture and reached a plateau that lasted from the second to the eighth day. A peritoneal macrophage preparation was co-cultured on overlaying collagen gel containing EC. The macrophage preparation increased tube length from the second to the fourth day in a time-dependent manner. IFN-γ (2.2 and 6.5 ng/ml) enhanced the effect of co-cultured macrophages from the fourth to the eighth day. The coditioned media derived from macrophages after 4 and 6 days of exposure to IFN-γ (6.5 ng/ml) also showed significantly enhanced tube formation induced by macrophage-conditioned medium. However, IFN-γ (6.5 ng/ml) did not influence the activity of the macrophage-conditioned medium. These results suggest that IFN-γ enhances angiogenesis from EC of rat aorta by releasing an angiogenic factor from macrophages.     

Vessel pruning or healing: endothelial metabolism as a novel target?

Introduction: Antiangiogenic drugs were originally designed to starve tumors by cutting off their vascular supply. Unfortunately, when these agents are used as monotherapy or in combination with chemotherapy, they provide only modest survival benefits in the order of weeks to months in most cancer patients. Strategies normalizing the disorganized tumor vasculature offer the potential to increase tumor perfusion and oxygenation, and to improve the efficacy of radio-, chemo- and immunotherapy, while reducing metastasis.

Areas covered: This review discusses tumor vascular normalization (TVN) as an alternative strategy for anti-angiogenic cancer treatment. We summarize (pre)-clinical strategies that have been developed to normalize tumor vessels as well as their potential to enhance standard therapy. Notably, we describe how targeting endothelial cell metabolism offers new possibilities for antiangiogenic therapy through evoking TVN.

Expert opinion: Several drugs targeting VEGF signaling are now clinically used for antiangiogenic cancer treatment. However, excessive blood vessel pruning impedes perfusion and causes tumor hypoxia, known to promote cancer cell dissemination and impair radio-, chemo- and immunotherapy. Normalized vessels lessen tumor hypoxia, impair cancer cell intravasation and enhance anticancer treatment. New data indicate that targeting endothelial cell metabolism is an alternative strategy of antiangiogenic cancer treatment via promotion of TVN.     

Protective effects of Ginkgo biloba extract against lysophosphatidylcholine-induced vascular endothelial cell damage

ＰｒｏｔｅｃｔｉｖｅｅｆｅｃｔｓｏｆＧｉｎｋｇｏｂｉｌｏｂａｅｘｔｒａｃｔａｇａｉｎｓｔｌｙｓｏｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅｉｎｄｕｃｅｄｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｃｅｌｄａｍａｇｅＣＨＥＮＪｉａｎＸｉｏｎｇ，ＣＨＥＮＷｅｉ...     

Oxidized low-density lipoprotein and β-glycerophosphate synergistically induce endothelial progenitor cell ossification

Aim:

To investigate the ability of ox-LDL to induce ossification of endothelial progenitor cells (EPCs) in vitro and explored whether oxidative stress, especially hypoxia inducible factor-1α (HIF-1α) and reactive oxygen species (ROS), participate in the ossific process.

Methods:

Rat bone marrow-derived endothelial progenitor cells (BMEPCs) were cultured in endothelial growth medium supplemented with VEGF (40 ng/mL) and bFGF (10 ng/mL). The cells were treated with oxidized low-density lipoprotein (ox-LDL, 5 μg/mL) and/or β-glycerophosphate (β-GP, 10 mmol/L). Calcium content and Von Kossa staining were used as the measures of calcium deposition. Ossific gene expression was determined using RT-PCR. The expression of osteocalcin (OCN) was detected with immunofluorescence. Alkaline phosphatase (ALP) activity was analyzed using colorimetric assay. Intercellular reactive oxygen species (ROS) were measured with flow cytometry.

Results:

BMEPCs exhibited a spindle-like shape. The percentage of cells that expressed the cell markers of EPCs CD34, CD133 and kinase insert domain-containing receptor (KDR) were 46.2%±5.8%, 23.5%±4.0% and 74.3%±8.8%, respectively. Among the total cells, 78.3%±4.2% were stained with endothelial-specific fluorescence. Treatment of BMEPCs with ox-LDL significantly promoted calcium deposition, which was further significantly enhanced by co-treatment with β-GP. The same treatments significantly increased the gene expression of core-binding factor a-1 (cbfa-1) and OCN, while decreased the gene expression of osteoprotegerin (OPG). The treatments also significantly enhanced the activity of ALP, but did not affect the number of OCN⁺ cells. Furthermore, the treatments significantly increased ROS and activated the hypoxia inducible factor-1α (HIF-1α). In all these effects, ox-LDL acted synergistically with β-GP.

Conclusion:

Ox-LDL and β-GP synergistically induce ossification of BMEPCs, in which an oxidizing mechanism is involved.     

Comparative analysis of nanosystems’ effects on human endothelial and monocytic cell functions

Abstract

The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0–400 µg/mL) on endothelial-monocytic cell interactions in response to TNF-α was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0–100 µg/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0–400 µg/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25–50 µg/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60–70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.     

Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain Ⅲ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain III (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [^3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells. RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. YcomlD3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Inhibitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro

AIM: To investigate the role of quercetin (Que) in the proliferation of cultured human skin microvascular endothelial cells (MVEC). METHODS: Cell count and [methyl-~3H]thymidine ([~3H]TdR) uptake assay were used to measure the effect of Que in the proliferation of cultured MVEC. Cytotoxicity of Que on MVEC was also evaluated by ~(51)Cr release assay. RESULTS: When MVEC were treated with Que, the proliferation was significantly inhibited in a time-course and dose-dependent manner. Que 5 μmol/L did not inhibit the proliferation of MVEC. When the concentration of Que increased to 20, 40, 80, and 160μmol/L, the cell numbers per well were decreased and the inhibition rate was 12.2％, 23.5％, 35.3％, and 54.1％ respectively with IC_(50) of 138 μmol/L. The inhibitory rate of [~3H]-TdR uptake was 18.7％, 34.4％, 48.9％, and 62.5％ respectively (IC_(50)=87.5 μmol/L). ~(51)Cr release assay showed that Que 160μmol/L incubated with MVEC from 1 to 16h had no clear cytotoxicity compared with control group. CONCL