Toxicity of cooking oil fume derived particulate matter: Vitamin D3 protects tubule formation activation in human umbilical vein endothelial cells.

Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) is the main source of indoor pollution. Exposure to COFs-derived PM2.5 can cause oxidative stress and affect angiogenesis. Here we investigated the roles of vitamin D3 (VD3) in protecting tubule formation injury induced by COFs-derived PM2.5, and the roles of ROS/NLRP3/VEGF signaling pathway in the effects. Human umbilical vein endothelial cells (HUVECs) were exposed to 0 (1‰ DMSO), 1000 nmol/l VD3, 100 µg/ml PM2.5, and 1000 nmol/l VD3 + 100 µg/ml PM2.5, respectively. Cell viability and tube formation, as well as protein and mRNA levels were measured. The results showed that exposure of COFs-derived PM2.5 dose-and time-dependently reduced the viability of HUVECs, increased the levels of mitochondrial and intracellular ROS, and changed the mitochondrial membrane potential level. While co-incubation with VD3 rescued these adverse effects. Both Western blot and real-time PCR (RT-PCR) showed that the expressions of NLRP3, caspase-1, Interleukin (IL)-1ß, and IL-18 in COFs-derived PM2.5 exposure group increased significantly, which could be effectively decreased by co-incubation with VD3. COFs-derived PM2.5 exposure could also reduce the expression of VEGF, while co-incubating HUVECs with VD3 evidently up-regulated the protein level of VEGF in HUVECs. In addition, COFs-derived PM2.5 could also inhibit the tube formation of HUVECs in vitro, which could be effectively rescued by the co-incubation of VD3. Our study proved that COFs-derived PM2.5 could damage the tubule formation of HUVECs in vitro, which could be effectively rescue by co-incubation with VD3, in which processes the ROS/NLRP3/VEGF signaling pathway played a crucial role. It provides a new theoretical basis for further study on the toxicity of PM2.5 to umbilical cord blood vessels.

Ductus Venosus Agenesis as a Marker of Pallister-Killian Syndrome.

The ductus venosus (DV) is a shunt that allows the direct flow of well-oxygenated blood from the umbilical vein (UV) to the coronary and cerebral circulation through the foramen ovale. Its agenesis has been associated with chromosomal abnormalities and rare genetic syndromes, structural defects, intrauterine growth restriction (IUGR) and even antepartum fetal demise. Pallister-Killian Syndrome (PKS) is a rare sporadic disorder with specific tissue mosaic distribution of an extra 12p isochromosome (i(12p)). Its main clinical features are moderate to severe intellectual disability/neuromotor delay, skin pigmentation abnormalities, typical facial appearance, variable association with multiple congenital malformations and epilepsy. Though prenatal findings (including congenital diaphragmatic hernia, ventriculomegaly, congenital heart disease, polyhydramnios, and rhizomelic shortening) have been described in literature, prenatal diagnosis is difficult as there are no associated identification signs no distinctive or pathognomonic signs, and some of these malformations are hard to identify prenatally. The tissue mosaicism linked to this syndrome and the decrease of the abnormal clone carrier of the i(p12) after successive trypsinizations of cultured cells makes the diagnosis even more challenging. We present the case of a 27.5 weeks pregnant woman with a fetal ductus venosus agenesis (DVA) as the main guide marker. To our knowledge this is the first case published in literature reporting a DVA as a guide sign to diagnose a complex condition as Pallister-Killian syndrome. We also underscore the key role of new genetic techniques as microarrays to avoid misdiagnosis when only a subtle sonographic sign is present in complex conditions like this.

The Role of Maternal Gestational Diabetes in Inducing Fetal Endothelial Dysfunction.

Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5 mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real-time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2',7' Dichlorodihydrofluorescein Diacetate (DCFH-DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC versus c.HUVEC (>1.5-fold change; P < 0.05). Functional clustering analysis of these differentially expressed genes revealed participation in inflammatory responses (including adhesion) which may be related to pathological outcomes. Of these genes, ICAM-1 was validated as upregulated, confirming microarray results. Additional confirmatory immunofluorescence staining revealed increased protein expression of ICAM-1 compared with c.HUVEC which was reduced by vitamin C treatment (100 µM). Thus, maternal diabetes induces persistent alterations in fetal endothelial function and gene expression following glucose normalization and antioxidant treatment could help reverse endothelium dysfunction.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells.

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown-knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

Tendon tissue engineering using cell-seeded umbilical veins cultured in a mechanical stimulator.

The goal of this study was to investigate the effect of cyclic mechanical stimulation on mesenchymal stem cells (MSCs) seeded within human umbilical veins (HUVs), and to determine the potential of the engineered constructs to function as tendon tissue replacement models. Decellularized HUVs were seeded with MSCs embedded in type I collagen hydrogel. A mechanical stimulator for tissue engineering applications was specifically designed to cyclically tension the constructs for durations up to 2 weeks, where controls were left untensioned. This HUV model system seeded with a cellular collagen gel, coupled with mechanical stimulation, resulted in improved mechanical properties compared to other tendon tissue engineered constructs composed of cellular collagen gel alone, without any additional supporting scaffold. After 2 weeks of culture an increase in cell number was measured for both tensioned and untensioned constructs; however, the increase was at least eightfold higher for stimulated samples. Microscopically, cyclically tensioned samples showed parallel orientation of collagen fibers and spindle-shaped cell nuclei mimicking the morphology of native tendons. Moreover, mechanostimulation resulted in significantly stronger (156%) and stiffer (109%) constructs compared to untensioned samples. This engineered tendon model had an ultimate tensile strength value only one order of magnitude lower than human tendons and strain values in the range of human tendons. The results documented are promising and can be further improved by optimizing potentially critical culture parameters such as seeding density, loading regimes, and mechanostimulation durations.

Metronomic chemotherapy dosing-schedules with estramustine and temozolomide act synergistically with anti-VEGFR-2 antibody to cause inhibition of human umbilical venous endothelial cell growth.

UNLABELLED: The effects of 'metronomic' or extended chemotherapy dosing schedules (ECS) are mediated through poorly understood anti-angiogenic mechanisms. ECS combined with biological anti-angiogenic agents have produced promising pre-clinical results. MATERIALS AND METHODS: We have expanded the list of agents with an in vitro ECS profile to include the methylating agent temozolomide (Temodal) and the anti-mitotic agent estramustine (Estracyt). These agents were also combined with a specific anti-angiogenic inhibitor IMC-1C11 and a non-specific agent with anti-angiogenic properties, Compound 5h. The in vitro HUVEC ECS model system was optimised and cell proliferation assays undertaken. RESULTS: As a single agent, estramustine inhibited endothelial cell proliferation with an IC50 of 4.5 microM and was active at 10-33% of the maximum tolerated dose (MTD) from clinical schedules, whilst temozolomide had IC50 of 6.6 microM and was active at 1-6% of MTD. In combination, significant synergy was seen with IMC-1C11 in combination with either drug, whilst modest additive effects were observed with Compound 5h. None of the combinations resulted in significant cytotoxicity or apoptosis. DISCUSSION: The results show that ECS of temozolomide and estramustine can be significantly enhanced when combined with specific anti-angiogenic inhibitors in an in vitro HUVEC system.

Subcellular topological effect of particle monolayers on cell shapes and functions.

We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene-co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell-cell adhesion became stronger than cell-substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm(-2), some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and the response was dependent on the pitch of particle monolayers.

Annexin A3 is a potential angiogenic mediator.

Angiogenesis is a complex process that is regulated by a variety of angiogenic activators and inhibitors. Disruption of the balanced angiogenesis leads to the progress of diseases such as tumor growth, rheumatoid arthritis, and various blood vessel-related disorders. Even though a number of proteins involved in angiogenesis have been identified so far, more protein factors remain to be identified due to complexity of the process. Here we report that annexin A3 (ANXA3) induces migration and tube formation of human umbilical vein endothelial cells. High level of vascular endothelial growth factor (VEGF), a prominent angiogenic factor, is also detected in conditioned medium obtained from cells transfected with ANXA3 expression plasmid. Reporter assays show that ANXA3 enhances hypoxia-inducible factor-1 (HIF-1) transactivation activity. Taken together, our results suggest that ANXA3 is a novel angiogenic factor that induces VEGF production through the HIF-1 pathway.

Bile leakage during transjugular intrahepatic portosystemic shunt creation: in vitro effect of bile on growth and function of human umbilical vein endothelium.

PURPOSE: To evaluate the effect of bile on growth and proliferation of human umbilical vein endothelium cultured in vitro, with a view toward clarifying the effect of bile leakage during transjugular intrahepatic portosystemic shunt creation. MATERIALS AND METHODS: This study was approved by the ethical review committee, and written informed consent was obtained from all mothers. Endothelial cells (ECs) were collected from human umbilical veins and cultured in vitro. After 24-48 hours in culture, ECs were distributed into groups supplemented with the following concentrations of bile in the culture medium: 0%, 5.0%, 10.0%, 15.0%, 20.0%, and 25.0%. The cells were harvested 5 days after supplementation with bile. The morphologic features, von Willebrand factor (vWF) level, tetrazolium salt (MTT) assay value of light absorption, total protein level, and nitric oxide synthase (NOS) activity of the ECs were evaluated. RESULTS: All explanted cells were identified as ECs by using the vWF test. Compared with ECs in the control group without bile, ECs in culture medium with a bile concentration of 5.0%, 10.0%, or 15.0% showed no marked morphologic changes, whereas ECs in culture medium with a bile concentration of 20.0% or 25.0% were reduced greatly in number and looked markedly immature. The MTT value of light absorption, total protein level, and vWF secretion were significantly decreased (P < .05 for all) in ECs in culture medium with 25.0% bile compared with these parameters in ECs in culture medium without bile, although these parameters did not significantly differ between the ECs in culture medium of 5.0% or 10.0% bile and the ECs in culture medium without bile. Compared with NOS activity in ECs when no bile was present in the culture medium, NOS activity in ECs was significantly decreased at all bile concentrations (P < .05). CONCLUSION: Low concentrations of bile do not markedly inhibit cell growth; the inhibiting effect of bile on ECs progresses with an increase in bile concentration.

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth.

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.

Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells.

Contact-dependent interactions between endothelial cells (ECs), as well as between ECs and mural cells, play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have identified ephrinB2 and EphB4 as markers and makers of arteriovenous differentiation during vascular development. On the basis of these findings, we hypothesized that Ephephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions that provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts anti-adhesively and induces detachment of ECs, as demonstrated by (1) inhibition of adhesion to ephrinB2-Fc-coated culture dishes, (2) detachment of ECs grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, vascular endothelial growth factor (VEGF) gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is pro-adhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2-mediated repulsive signals are transduced by EphB4, as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4-overexpressing ECs. Confrontation experiments of EphB4-overexpressing ECs with ECs overexpressing full-length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, these data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.

Intermediate filament proteins in developing human arteries.

The distribution of intermediate filament proteins in adult human blood vessels and in human fetal elastic arteries is relatively well-known. However, the distribution of these proteins in the course from neonate to adult has not been established. In this investigation, human postnatal arteries were studied with immunohistochemistry, using antibodies targeted on the intermediate filament proteins desmin, vimentin and cytokeratins 8, 18 and 19. Vimentin was present in most smooth muscle cells in all vessels and at all ages. The proportions of desmin-expressing cells increased in the elastic arteries during the first year of life and was higher in the pulmonary trunk than in the aorta. In the muscular arteries, the proportion of desmin-labelled cells increased in the coronary and the deep femoral arteries, but remained constant in the renal and the cerebral arteries. Cytokeratins were detected in the pulmonary trunk earlier than in the aorta. Cytokeratins were present throughout the wall of the ductus arteriosus, but desmin was present only in some cells. Thus, there are postnatal changes in the distribution of intermediate filament proteins in the elastic arteries and in some muscular arteries, whereas the intermediate filament pattern remains unchanged in other muscular arteries.

Characteristics of vascular wall cells subjected to dynamic cyclic strain and fluid shear conditions in vitro.

We recently developed an in vitro silicone rubber tubular apparatus, the vascular simulating device (VSD), which simulates pressure, flow, and strain characteristics of peripheral arteries (Benbrahim et al., 1994, J. Vasc. Surg. 20, 184-194). In this report, we tested the ability of silicone rubber surfaces to support the growth and differentiation of endothelial cells (EC) and smooth muscle cells (SMC) and studied the effects of arterial levels of pressure, flow, and strain on these properties. Human umbilical and saphenous vein EC and bovine aortic EC and SMC were cultured on coated and uncoated silicone rubber in flat and tubular configurations (6 mm inner diameter) and on tissue culture plastic (TCP). Attachment, growth, and differentiation were compared on these surfaces. In addition, the effects of arterial pressure, flow, and strain conditions on adhesion and subsequent growth and differentiation were studied in the tubular configuration. Attachment and growth of vascular wall cells on fibronectin-coated silicone rubber was similar to that obtained on TCP. Application of arterial levels of pressure, flow, and strain did not alter adhesion of the cells to the tubes. Subsequent passage of these cells demonstrated that attachment, growth, and differentiation (uptake of LDL and expression of factor VIII-related antigen by EC and expression of muscle-specific actin by SMC) were similar in cells derived from experimental and control tubes which were not subjected to arterial conditions. Finally, mRNA expression of specific "housekeeping" genes was similar in cells isolated from experimental and control tubes. We conclude that the VSD supports the culture of viable and differentiated EC and SMC. These experiments demonstrate that it is possible to evaluate the effects of arterial strain and fluid shear on vascular wall cells in vitro, in a configuration similar to the blood vessel wall.

A study of the ultrastructure of developing human umbilical vessels.

Electron microscopic techniques were used to examine the ultrastructure of developing human umbilical arteries and vein (8-12, 13-17 and 37-40 wk gestational age). These showed that with increasing age there is (1) an increase in the size of the lumen and the thickness of the media; (2) an increase in the ratio of contractile smooth muscle phenotypic cells; (3) an increase in the myofilament content of the smooth muscle cells and the number of Weibel-Palade bodies; (4) a decrease in the glycogen content; (5) an appearance of microvilli on the luminal surface of the endothelium. Lipid vesicles, nerves and vasa vasorum were not observed in any region of the umbilical vein or arteries.

Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics.

The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1 beta and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na(+)-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1 beta and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.

Stimulation of endothelial cell migration in culture by ladsin, a laminin-5-like cell adhesion protein.

Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin alpha 3 beta 1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.