Presence Of Endothelial Cells Research Articles

Hmga2 regulates self-renewal of retinal progenitors.

In vertebrate retina, histogenesis occurs over an extended period. To sustain the temporal generation of diverse cell types, retinal progenitor cells (RPCs) must self-renew. However, self-renewal and regulation of RPCs remain poorly understood. Here, we demonstrate that cell-extrinsic factors coordinate with the epigenetic regulator high-mobility group AT-hook 2 (Hmga2) to regulate self-renewal of late retinal progenitor cells (RPCs). We observed that a small subset of RPCs was capable of clonal propagation and retained multipotentiality of parents in the presence of endothelial cells (ECs), known self-renewal regulators in various stem cell niches. The self-renewing effects, also observed in vivo, involve multiple intercellular signaling pathways, engaging Hmga2. As progenitors exhaust during retinal development, expression of Hmga2 progressively decreases. Analyses of Hmga2-expression perturbation, in vitro and in vivo, revealed that Hmga2 functionally helps to mediate cell-extrinsic influences on late-retinal progenitor self-renewal. Our results provide a framework for integrating the diverse intercellular influences elicited by epigenetic regulators for self-renewal in a dynamic stem cell niche: the developing vertebrate retina.

Abstract LB-146: Investigation of Gas6/Axl signaling in clear cell renal cell carcinoma tumor cells and endothelial cells in presence of the first line treatment drug Sunitinib Malate

Abstract Introductory sentence: The aim is to investigate whether Gas6 dependent Axl activation and downstream signaling can occur in clear cell Renal Cell Carcinoma tumor cells (ccRCCs) and Endothelial cells (ECs) during treatment with Sunitinib Malate. Experimental set up: The ccRCC cell line 786-O and ECs of human aortic origin (HAECs) were grown in vitro, serum-starved over night and pre-incubated with Sunitinib Malate for 2 hours prior 100 ng/mL Gas6 stimulation for 10 minutes. Activation of Axl in terms of induced tyrosine phosphorylation was determined by immunoprecipitation and immunoblotting. Gas6-induced downstream signaling of Axl was analyzed by immunoblotting for phosphorylation of Akt and Erk protein in total cell lysates. Gas6/Axl specific signaling was verified by pre-incubation with an Axl specific kinase inhibitor, R428. Functionality of Sunitinib Malate was verified by inhibition of VEGFR-activation in ECs. In addition, prolonged stimulation of ccRCCs with Gas6 only was analyzed up to 2 days. Longterm effects on induced Axl activation and downstream Akt and Erk signaling in ccRCCs were investigated as described above. Results: In ccRCCs and ECs, Gas6-induced Axl activation and downstream signaling are resistant to physiologically relevant and higher doses of Sunitinib Malate. Gas6/Axl-signaling activates Akt and Erk pathways in presence of Sunitinib Malate. Sunitinib Malate contributes to Gas6 signaling with an increased phospho-Axl, Akt and Erk activation in ccRCCs and ECs. Axl is constitutively activated in ccRCCs upon longterm Gas6 stimulation, with early peak Erk activation and sustained Akt activation. Conclusions and importance: RCC is a lethal cancer with bad prognosis due to development of chemoresistance and recurrence of a more aggressive tumor. The Gas6/Axl system influences many cellular processes, including survival, migration and invasion, contributing to both chemoresistance and metastasis of several tumor types. Axl overexpression is also associated with poor patient outcome. Our data indicates that the Gas6/Axl system can be constitutively activated in ccRCCs with sustained Axl activation and downstream signaling of pathways known to apply survival, growth and proliferative advantage to tumor cells. Sunitinib Malate, first-line anti-angiogenic targeted therapy drug used in advanced RCC, does not inhibit and moreover, increases Gas6/Axl signaling in both ccRCCs and ECs. Importantly this suggests that, Sunitinib Malate, while targeting ECs and tumor-angiogenesis, simultaneously possibly provide a pro-tumorigenic tumor microenvironment where Gas6/Axl signaling, not inhibited by Sunitinib Malate, stimulates ccRCCs and ECs more intensively than in the non-treated tumor microenvironment. Altogether, Gas6/Axl signaling during targeted therapy is possibly contributing to the acquired chemoresistance and recurrence of aggressive tumors in patients treated for advanced RCC. Citation Format: Anna Gustafsson, Björn Dahlbäck. Investigation of Gas6/Axl signaling in clear cell renal cell carcinoma tumor cells and endothelial cells in presence of the first line treatment drug Sunitinib Malate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-146. doi:10.1158/1538-7445.AM2014-LB-146

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells?

PurposeNumerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. Material/methodsAfter incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. ResultsLower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1μg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10μg gallic acid equivalent/ml. ConclusionsThe results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering

Liver tissue engineering requires a suitable cell source, methodologies to assemble the cells within their niche microenvironments in a spatially defined manner, and vascularization of the construct in vivo for maintenance of hepatocyte viability and function. Recently, we have developed methods of encapsulating cells within separate domains in multi-component hydrogel fibers and methods of assembling fibers to form 3D-patterned tissue constructs. In the present work, we have combined these approaches to encapsulate hepatocytes and endothelial cells within their specific niches, and to assemble them into endothelialized liver tissue constructs. The hepatocytes and endothelial cells were obtained in parallel by differentiating human recombinant protein-induced human pluripotent stem cells, resulting in a construct which contained genetically identical endothelial and parenchymal elements. We were able to demonstrate that the presence of endothelial cells in the scaffold significantly improved hepatocyte function in vitro and facilitated vascularization of the scaffold when implanted in a mouse partial hepatectomy model. The in vivo studies further asserted that integration of the scaffold with host vasculature had occurred, as demonstrated by the presence of human albumin in the mouse serum.

Seeing the Inner from Outer

Since Furchgott et al1 showed that acetylcholine requires the presence of endothelial cells to induce vasodilation, the importance of the endothelial cell layer for vascular homeostasis has been increasingly appreciated. Endothelial dysfunction was initially identified as impaired vasodilation to specific stimuli such as acetylcholine or bradykinin. A broader understanding of the term would include not only reduced vasodilation but also a proinflammatory and prothrombic state associated with dysfunction of the endothelium.2 Figure 1 describe regulation function of endothelium during normal and dysfunction condition.3 In human, endothelial dysfuntion was first described in forearm of hypertensive patient.4 Dysfunction of the endothelium has been attributed to the pathophysiology of different forms of cardiovascular disease, including hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure.2

BASCing in the Presence of Endothelial Cells

Lung stem cells receive signals from endothelial cells about when to differentiate.

Endothelial Cells Modulate Platelet Response to Dynamic Shear Stress

The goal of this study was to investigate how endothelial cells affect platelet response to physiologically relevant dynamic shear stress. In a cone and plate shearing device, washed platelets were exposed to constant (0.1, 0.3, 1, and 3 Pa) or dynamic (normal, low and temporally elevated) shear stress waveforms with or without the presence of endothelial cells. Platelet surface CD62P, CD42b and CD41a expression was measured using flow cytometry. Endothelial cell surface ICAM-1 and vWF expression was measured using ELISA, endothelial cell surface PECAM-1 expression was measured using fluorescence microscopy and endothelial cell derived microparticle generation was measured using flow cytometry. Elevated constant shear stress significantly increased platelet surface CD62P expression, and reduced platelet surface CD42b and CD41a expression. However, under dynamic flow conditions, low or elevated shear stress did not cause significant changes in platelets. But with the presence of endothelial cells, dynamic shear stress induced a significant increase in platelet surface CD62P expression and reduced platelet surface CD42b and CD41a expression. A significant increase in soluble vWF and the number of endothelial cell microparticles was observed, suggesting that soluble vWF and endothelial microparticle may be the mediators that induced platelet activation under pathological dynamic flow. These results suggest that pathological dynamic shear stress may not activate platelets directly. Through shear stress stimulated endothelial cells, platelets may become activated. As a protective mechanism, following cell activation, platelet adhesion protein expression is reduced to restrain platelet adhesion, aggregation and thrombus formation.

Immunomagnetic exclusion of PECAM-1-positive endothelial cells in fetal mouse liver cell cultures causes impaired growth and gene expression of hepatoblasts and stellate cells.

Previous studies using mice having defective VEGF signaling have demonstrated that vascular development is indispensable for early hepatic organogenesis. However, not only whether its action lasts during later hepatic development, but also what molecules are involved in that action remains to be determined. The present study was undertaken to examine the effects of primitive sinusoidal endothelial cells on hepatic growth and maturation in primary culture of fetal mouse liver cells, and to determine their molecular mechanisms. When endothelial cells were excluded from E12.5 liver cell cultures by using PECAM-1-antibody-coated magnetic beads, the growth of hepatoblasts and stellate cells was conspicuously reduced and hepatic maturation was also suppressed. Conditioned medium prepared from fetal liver cell cultures containing almost all hepatic cell types stimulated the growth and gene expression of hepatoblasts and stellate cells similarly to the cultures in the presence of endothelial cells. HGF mRNA expression was downregulated in endothelial cellfree cultures of fetal liver cells, and the addition of HGF to the culture medium rescued the cells from the effects of endothelial cell depletion. These data suggest that humoral factors, including HGF, which are produced by endothelial cells or stellate cells, are involved in fetal hepatocyte growth and maturation.

Epithelial-to-mesenchymal transition and slit function of mesothelial cells are regulated by the cross talk between mesothelial cells and endothelial cells

Peritoneal dysfunction is a major factor leading to treatment failure of peritoneal dialysis (PD). However, the precise mechanism of the peritoneal diffusion changes related to PD remains to be elucidated. To this end, we have established a novel peritoneal diffusion model in vitro, which consists of a three-dimensional culture system using a collagen vitrigel membrane chamber and a fluid-stream generation system. This artificial peritoneal model revealed that high-glucose culture medium and fluid flow stress promoted the epithelial-mesenchymal transition (EMT) process of mesothelial cells and that endothelial cells inhibited this mesothelial EMT process. Mesothelial cells in the EMT state showed high expression of connective tissue growth factor and low expression of bone morphogenic protein-7, while non-EMT mesothelial cells showed the opposite expression pattern of these two proteins. In addition, these protein expressions were dependent on the presence of endothelial cells in the model. Our model revealed that the endothelial slit function was predominantly dependent on the covering surface area, while the mesothelial layer possessed a specific barrier function for small solutes independently of the surface area. Notably, a synergic barrier effect of mesothelial cells and endothelial cells was present with low-glucose pretreatment, but high-glucose pretreatment abolished this synergic effect. These findings suggest that the mesothelial slit function is not only regulated by the high-glucose-induced EMT process but is also affected by an endothelial paracrine effect. This peritoneal diffusion model could be a promising tool for the development of PD.

Two Ways to Make Great Contributions

This commentary is about 2 investigators, Robert Furchgott and Stanton Glantz, who have written some of the most highly cited articles in Circulation Research . Furchgott and Glantz changed our scientific world and our society. As I read the list of most highly cited articles in the 60 years of publication of Circulation Research , 2 stories came to mind. The first story is of a great discovery, and the second is of a great contribution to reduction of cardiovascular disease. First is Robert Furchgott’s finding that endothelium is critical in normal vasomotor function of arteries. His finding was followed by a series of studies that demonstrated the importance of endothelial dysfunction in atherosclerosis, hypertension, and other diseases. Second is a story about Stanton Glantz, who made major contributions to our understanding of ventricular diastolic function, which foreshadowed our understanding of diastolic heart failure. However, for me, the truly inspiring story is how Glantz successfully challenged the tobacco industry, with enormous consequences for cardiovascular health. Furchgott’s1 Brief Review, which is the article in Circulation Research with the third greatest number of citations, begins in his modest, understated manner: “A few years ago, we discovered that the relaxation by acetycholine of isolated preparations of arteries is strictly dependent on the presence of endothelial cells.” Several years before the Brief Review was published, Paul Vanhoutte had invited Furchgott to a small meeting to talk about his finding, which had been presented as an abstract. I talked with Furchgott, and thought that his finding was interesting, but not likely to be of great importance. I reasoned that there is not much acetylcholine in blood vessels, or in blood to which endothelium is exposed and, therefore, it was not likely that this vasorelaxing effect of acetylcholine in the aorta ex vivo was of …

200 NEUTROPHIL-DERIVED MICROPARTICLES MODULATE MONOCYTE MIGRATION IN AN ENDOTHELIAL DEPENDENT MANNER

<h3>Introduction</h3> Neutrophil depletion can delay atherogenesis and conversely increasing circulating neutrophils enhances plaque progression in mice. Lack of evidence for the presence of neutrophils in atherosclerotic plaques makes their role in disease progression less clear. Neutrophils may have a greater role in facilitating the movement of other cells into the vessel wall, possibly through the release of microparticles. The ability of microparticles to deliver cargo to target cells may be an explanation for neutrophil-specific markers being co-localised with endothelial cells in atherosclerotic plaques, despite the absence of neutrophils themselves. <h3>Methods</h3> Neutrophils were incubated with PBS, fMLP (10–5 M), and acetylated LDL (AcLDL, 20 µg/ml) to stimulate microparticle formation. To remove residual fMLP, microparticle suspensions were dialysed. Microparticles were characterised and quantified using a standardised flow cytometry method. Their adhesion to human coronary artery endothelial cells (hCAEC) after incubation with anti-ICAM-1 or isotype control was quantified using flow cytometry. Internalisation was visualised using confocal microscopy. Microparticle binding to and activation of monocytes was also investigated using flow cytometry. Monocytes migration to CCL-2 in the presence of neutrophil-derived microparticles with and without hCAEC was also investigated. <h3>Results</h3> Neutrophil-derived microparticles adhered to hCAEC in an ICAM-1 dependent manner and were internalised. Microparticles bound to monocytes regardless of stimuli, but their adhesion was lower than observed with hCAEC. Only microparticles from neutrophils stimulated with fMLP induced monocyte L-selectin shedding and significantly enhanced monocyte-hCAEC adhesion. Microparticles increased monocyte migration to CCL2 in an endothelial cell and CD18-dependent manner. <h3>Conclusions</h3> Microparticles bind to ICAM-1 on the surface of hCAEC and are internalised. Monocyte migration is enhanced by neutrophil-derived microparticles only in the presence of endothelial cells. In conclusion, neutrophil-derived microparticles may play a role in activating hCAEC, potentially by their uptake, and thus amplify inflammatory responses at sites of atherosclerotic lesion development.

Abstract B91: A novel role for the secreted growth factor progranulin in angiogenesis

Abstract Progranulin (also called PC cell-derived growth factor, acrogranin or proepithelin), is a secreted glycoprotein with growth factor-like activities. It is often highly over-expressed in tumors including cancers of the breast, ovary, endometrium, kidney, prostate, bladder and liver. It stimulates cellular proliferation, migration and survival and increases the growth of cancers in mice. Recent work suggests that progranulin has, in addition, potentially important roles in fibroblast activation during the formation of tumor stroma. An elevated progranulin level has been observed in endothelial cells in some cancers including ovarian tumors, esophageal carcinomas and gliomas. Since progranulin is not expressed in normal quiescent endothelia, its presence in endothelial cells from cancers prompted us to propose that progranulin may have a novel role in tumor angiogenesis. It is, however, unknown at present, how, if at all, progranulin modulates angiogenesis in vivo. To address this question we created transgenic models in which progranulin expression is specifically targeted to endothelial cells under the control of the Tie2 promoter. Three independent transgenic lines were obtained, with lower (Tie2-GrnLo), intermediate (Tie2-GrnMid) and higher (Tie2-GrnHi) transgene copy number. Increased mortality was observed in heterozygotic crosses from all three transgenic lines, with the highest mortality and lowest germline transmission ratio in the Tie2-GrnHi line; at three weeks after birth the germline transmission ratios of Tie2-Grn positive mice were 26.01%, 19.61% and 4.72% for PgrnLo, PgrnMid and PgrnHi respectively. Premature deaths occurred during the perinatal period after embryonic day 17.5 and before post-natal day 3. Importantly, we observed an array of vascular abnormalities in Tie2-Grn transgenic neonates. These include extensive bleeding into body cavities such as the pericardial space as well as smaller localized hemorrhages in multiple organs. The abnormal blood vessels had dramatically enlarged diameters with irregular edges; several had an incomplete basement membranes and, typically, the perivascular cell coverage was reduced or absent when compared to Tie2-Grn negative litter mates. To establish the developmental timing of vascular disruption, we examined Tie2-GrnHi mice from embryonic day 10.5 to fetal age 17.5. An apparently normal primitive vasculature was established at E10.5 in Tie2-Grn positive embryos suggesting that vasculogenesis proceeded normally. The earliest onset of vascular impairment was observed at E15.5, after vasculogenesis has occurred but during active angiogenesis. As with neonates, from E15.5 and later, vessels became enlarged and perivascular cell investiture was reduced contributing to loss of structural integrity and causing the blood vessels to be prone to hemorrhaging. Given the vessel dilation in Tie2-Grn positive late-stage fetuses and neonates we conclude that progranulin is a novel regulator of angiogenesis. The elevated expression of progranulin by endothelial cells is observed in some cancers in vivo, and, as we report here, in transgenic mice results in the development of enlarged, disorganized, thin-walled vessels that resemble to some extent the disorganized blood vessels in tumors. We suggest, therefore, that progranulin may be significant in regulating tumor angiogenesis. Citation Format: Huishi Toh, Ming Cao, Yonghua Zhang, Eugene Daniels, Andrew Bateman. A novel role for the secreted growth factor progranulin in angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B91.

Influence of pressurized cyclic stretch and endothelial cell presence on multipotent stem cell osteogenic commitment

Applied mechanical stretch and blood vessel invasion are key stimuli to which progenitor cells are exposed in post-natal endochondral bone formation. Understanding the combined effects of cyclic stretch and endothelial cell (EC) presence on multipotent stem cell (MSC) osteogenesis therefore has the potential to lead to improved MSC-based bone regeneration strategies. Toward this goal, 10T1/2 mouse MSCs were encapsulated in tubular poly(ethylene glycol) diacrylate [PEGDA] hydrogels with moduli within the "osteogenic" range in order to induce osteogenesis. Half of the constructs were fabricated with a luminal EC layer. All of the EC(+) (EC(+)/dyn(+)) and half of the EC(-) constructs (EC(-)/dyn(+)) were subjected to pressurized cyclic stretch in the absence of osteogenic media supplements, with remaining EC(-) constructs (EC(-)/dyn(-)) serving as static controls. At day 10 of culture, expression of the bone extracellular matrix protein osteopontin was over 3.3- and 1.9-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. At day 22 of culture, osteopontin levels could not be statistically distinguished from day 0 in the EC(+)/dyn(+) constructs and were one-third less than day 0 in the EC(-)/dyn(+) constructs. In contrast, at day 22 levels of an osteogenic marker alkaline phosphatase (AP) were over 2.4- and 1.4-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. Furthermore, at day 22 matrix mineralization in both dynamic groups was increased over 2.5-fold and over 9-fold relative to the EC(-)/dyn(-) and day 0 groups, respectively. Cumulatively, these results suggest that pressurized cyclic stretch alone significantly increases the rate/degree of osteogenesis relative to static culture. However, EC presence combined with pressured cyclic stretch appears to further enhance the rate/degree of MSC osteogenesis and/or to support a distinct osteogenic "fingerprint" compared to that promoted by cyclic stretch alone.

Surface Passivation of Carbon Nanoparticles with Branched Macromolecules Influences Near Infrared Bioimaging

A superior and commercially exploitable 'green synthesis' of optically active carbon nanoparticle (OCN) is revealed in this work. The naked carbon particles (<20 nm) were derived from commercial food grade honey. The fluorescence properties of these particles were significantly enhanced by utilizing hyberbranched polymer for surface passivation. A dramatic increase in near infrared emission was achieved compared to a linear polymer (PEG) coated carbon nanoparticles. Interestingly, as passivating agent becomes more extensively branched (pseudo generation 2 to 4), the average radiant efficiency amplifies considerably as a direct result of the increasing surface area available for light passivation. The particles showed negligible loss of cell viability in presence of endothelial cells in vitro. Preliminary in vivo experiment showed high contrast enhancement in auxiliary lymphnode in a mouse model. The exceptionally rapid lymphatic transport of these particles suggests that such an approach may offer greater convenience and reduced procedural expense, as well as improved surgical advantage as the patient is positioned on the table for easier resection.

Assessment of Protein C Anticoagulant Pathway by Thrombin Generation Assay in the Presence of Endothelial Cells

Abstract 3394The generation of thrombin is the pivotal event in the process of blood coagulation. In vivo, thrombin generation is regulated by cooperation between the vascular endothelium and the pro- and anti-coagulant systems in blood, such as the thrombin/endothelial cell-dependent activation of the protein C anticoagulant pathway that ultimately leads to inactivation of factors Va and VIIIa.In vitro, thrombin generation is assessed most commonly in platelet-poor plasma by a fluorogenic substrate-based thrombin generation assay (TGA) in microtiter plates. While this assay can accurately measure the kinetics of thrombin generation in plasma (including Lag time [LT], Peak thrombin [PT], and endogenous thrombin potential [ETP]), it does not assess the influence of the endothelial cell-dependent protein C pathway on thrombin generation. As a result, the assay has limited value in the assessment of the hypercoagulable patient.In the present study we introduced a surrogate endothelium to the TGA, thus including activated protein C-induced inactivation of factors Va and VIIIa in the assay system. Wells of flat-bottomed microtiter plates were coated with 3 × 104quiescent EA.hy926 endothelial-like cells which consistently express thrombomodulin (TM) as well as the endothelial protein C receptor (EPCR). The concentration of active TM associated with EA.hy926 in the assay well was determined by a chromogenic assay and found to be ∼0.5 nM. Tissue factor (TF)-initiated thrombin generation was evaluated in normal pooled plasma (NP), and in protein C-deficient (PCd), protein S-deficient (PSd), and heterozygous factor V Leiden (fVL) plasmas, in the presence or absence of endothelial cells.Thrombin generation in NP was reduced in the presence of endothelial cells as evidenced by a 55% reduction in PT, and a 40% reduction in ETP. LT was prolonged by 83% in the presence of endothelial cells. However, in PCd, PSd and fVL plasmas, endothelial-induced suppression of thrombin generation was blunted. Specifically, only relatively small reductions were observed in PT (26% in PCd, 28% in PSd, 20% in fVL) and ETP (12% in PCd, 6% in PSd, and 14% in fVL). Furthermore, only in the presence of endothelial cells, addition of protein C to PCd plasma dose-dependently reversed the effect of protein C deficiency on PT and ETP. In separate experiments in the absence of endothelial cells, we noted that while similar results were obtained in the presence of the soluble form of thrombomodulin, the concentration required (30 nM) was 60 times higher than that expressed on the endothelial cell monolayer. This suggests that protein C activation occurs more efficiently in the presence of EA.hy926 cells.In summary, by introducing an endothelial cell monolayer to the TGA, and measuring thrombin generation kinetics in the presence or absence of these cells, we have adapted the assay to assess the contribution of the protein C anticoagulant system to thrombin generation in a physiologically relevant manner. This novel approach not only enables the functions of the endothelial-dependent PC pathway by expressing TM and EPCR, but also may provide other endothelial components relevant to thrombin generation (such as tissue factor pathway inhibitor [TFPI]). This approach to thrombin generation assessment may therefore have both research and clinical applicability. Disclosures:No relevant conflicts of interest to declare.

Platelets From Sickle Cell Disease Individuals Induce Endothelial Activation, Demonstrating ICAM-1 and E-Selectin Adhesion Molecule Expression, Inflammatory Cytokine Production and Activation of NFκB Transcription Factor Gene Expression.

AbstractAbstract 2114 Introduction:Sickle cell disease (SCD) pathophysiology is associated with a hypercoagulable state that may contribute to the initiation and propagation of vaso-occlusion. Increased platelet activation has been described in SCD and SCD platelets may present augmented adhesion to the vascular endothelium, potentially contributing to vaso-occlusion. Aim:This study investigated whether platelets (PLTs) from SCD individuals are able to activate endothelial cells per se. Methods:Human umbilical vein endothelial cells (HUVEC) were cultured (1×106cells/well) on 6-well plates (37°C, 5% CO2). Subsequently, HUVEC were co-cultured in direct contact, or not, with washed PLTs (1×108PLTs/well) from healthy control individuals (CON, n=23) or steady-state SCD patients (SCD, n=47; 26 of which were on hydroxyurea therapy; 20mg/Kg/day) for 4h, 37°C, 5%CO2. After incubation, PLTs were removed; supernatants were reserved for cytokine quantification by ELISA, and HUVEC were analyzed by flow cytometry for CD62E (E-Selectin) and CD54 (ICAM-1) surface expression; gene expressions of ICAM1 and NFKBIA were analyzed by qPCR. Results:Basal ICAM-1 expression on the surface of HUVEC (39.6±3.2%, n=15) was significantly increased following their incubation in direct contact with SCD PLTs (46.1±3.1%, n=26, p<0.05, Wilcoxon test), but not CON PLTs (41.3±4.7%, n=12). E-selectin expression was also low level on the surface of HUVEC (0.9±0.2%, n=17), and was slightly but significantly increased following incubation of cells with SCD PLTs (6.1±2.2%, n=26, p<0.001), but not CON PLTs (3.6±1.5%, n=12, p>0.05). Repetition of these assays, but with the placement of transwell inserts in culture plates to separate the PLTs from HUVEC resulted in a 45% decrease in ICAM-1 expression (p<0.05), and 85% decrease in E-selectin (p<0.05) expression on the surface of HUVEC, following their incubation with SCD PLTs. HUVEC produce and release interleukin-8 (IL-8); basal IL-8 production by HUVEC (1×106cells/well) was 1.160±0.187ng/mL (n=21); this production was augmented in the presence of SCD PLTs (1.280±0.149ng/mL, n=42, p<0.01), but not by CON PLTs (1.127±0.157ng/mL, n=23). The influence of PLT IL-8 production on these values was negligible, as shown by data (not shown) demonstrating that PLT IL-8 production is low level and does not differ between CON and SCD PLTs. IL-1β is produced and released by PLTs (CON, 3.4±1.4ρg/mL, n=12; SCD, 6.5±1.5 ρg/mL, n=25, p>0.05), but this production was further increased when PLTs were co-cultured with HUVEC: SCD PLTs (10.3±4.2ρg/mL, n=47; p<0.01) and CON PLTs (5.8±2.4ρg/mL, n=25; p>0.05), compared to HUVEC alone (1.27±0.4ρg/mL, n=24). Gene expression of ICAM1 by HUVEC increased 6.3-fold in the presence of SCD PLTs (n=25, p<0.01), compared to basal expression (n=11), but was not altered in the presence of CON PLTs (n=11, p>0.05). The expression of the gene encoding the NFkB transcription factor, NKBIA, increased 3.4-fold in HUVEC following incubation with SCD PLTs (n=25, p<0.05), compared to basal NFKBIA expression (n=12); however NFKBIA expression in HUVEC was not significantly altered by CON PLTs (n=10, p>0.05). Conclusions:Results indicate that the contact of platelets, or products released from platelets, from patients with SCD may activate endothelial cells, in vitro, increasing adhesion molecule and IL-8 production, associated with an augmented expression of the gene encoding NFkB. Platelets produce IL-1β in greater quantities in the presence of endothelial cells, possibly contributing to endothelial cell activation; however the fact that transwell inserts significantly reduced SCD PLT-mediated endothelial activation indicates that the direct contact of PLTs (possibly via adhesion) is required for this activation. Data indicate that platelets adhered to vessel walls may play an important role in endothelial activation and, therefore, vaso-occlusive mechanisms in SCD. Disclosures:No relevant conflicts of interest to declare.

Radiation-induced galectin-1 by endothelial cells: a promising molecular target for preferential drug delivery to the tumor vasculature

The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature-targeting ligand, anginex, to improve the therapeutic ratio for strategies developed against solid tumors. Our findings indicate that galectin-1 is (a) one of the major receptors for anginex (b) overexpressed by tumor neovasculature and (c) further specifically upregulated in endothelial cells in response to radiation exposure as low as 0.5 Gy. An investigation of [18]-F-labeled anginex biodistribution in SCK tumors indicates that anginex is an effective targeting molecule for image and radiation-guided therapy of solid tumors. An anginex-conjugated liposome capable of being loaded with drug was shown to selectively target endothelial cells post-radiation. The presence of endothelial cells in a three-dimensional co-culture system with tumor cells developed to study tumor/endothelial cell interactions in vitro led to higher levels of galectin-1 and showed a further increase in expression upon radiation exposure when compared to tumor cell spheroids alone. Similar increase in galectin-1 was observed in tumor tissue originating from the tumor-endothelial cell spheroids in vivo and radiation exposure further induced galectin-1 in these tumors. The overall results suggest feasibility of using a clinical or subclinical radiation dose to increase expression of the galectin-1 receptor on the tumor microvasculature to promote delivery of therapeutics via the anginex peptide. This approach may reduce systemic toxicity, overcome drug resistance, and improve the therapeutic efficacy of conventional chemo/radiation strategies.

Angioneural Crosstalk in Scaffolds with Oriented Microchannels for Regenerative Spinal Cord Injury Repair

The aim of our work is to utilize the crosstalk between the vascular and the neuronal system to enhance directed neuritogenesis in uniaxial guidance scaffolds for the repair of spinal cord injury. In this study, we describe a method for angioneural regenerative engineering, i.e., for generating biodegradable scaffolds, produced by a combination of controlled freezing (freeze-casting) and lyophilization, which contain longitudinally oriented channels, and provide uniaxial directionality to support and guide neuritogenesis from neuronal cells in the presence of endothelial cells. The optimized scaffolds, composed of 2.5 % gelatin and 1 % genipin crosslinked, were characterized by an elastic modulus of ~51 kPa and longitudinal channels of ~50 μm diameter. The scaffolds support the growth of endothelial cells, undifferentiated or NGF-differentiated PC12 cells, and primary cultures of fetal chick forebrain neurons. The angioneural crosstalk, as generated by first forming endothelial cell monolayers in the scaffolds followed by injection of neuronal cells, leads to the outgrowth of long aligned neurites in the PC12/endothelial cell co-cultures also in the absence of exogenously added nerve growth factor. Neuritogenesis was not observed in the scaffolds in the absence of the endothelial cells. This methodology is a promising approach for neural tissue engineering and may be applicable for regenerative spinal cord injury repair.

Enhanced FcαRI‐mediated neutrophil migration towards tumour colonies in the presence of endothelial cells

Neutrophils potently kill tumour cells in the presence of anti-tumour antibodies in vitro. However, for in vivo targeting, the neutrophils need to extravasate from the circulation by passing through endothelial barriers. To study neutrophil migration in the presence of endothelial cells in vitro, we established a three-dimensional collagen culture in which SK-BR-3 tumour colonies were grown in the presence or absence of an endothelial barrier. We demonstrated that - in contrast to targeting FcγR on neutrophils with mAbs - targeting the immunoglobulin A Fc receptor (FcαRI) instead triggered neutrophil migration and degranulation leading to tumour destruction, which coincided with release of the pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α. Interestingly, neutrophil migration was enhanced in the presence of endothelial cells, which coincided with production of significant levels of the neutrophil chemokine IL-8. This supports the idea that stimulation of neutrophil FcαRI, but not FcγR, initiates cross-talk between neutrophils and endothelial cells, leading to enhanced neutrophil migration towards tumour colonies and subsequent tumour killing.

Preseeding of human vascular cells in decellularized bovine pericardium scaffold for tissue‐engineered heart valve: An in vitro and in vivo feasibility study

Human vascular cells from saphenous veins have been used for cell seeding on the synthetic scaffolds for constructing tissue-engineered heart valve (TEHV). However, little is known about the seeding of human vascular cells on bovine pericardium, a potential natural scaffold for TEHV. This study was aimed to assess the basic in vitro and in vivo characteristics of the human vascular cells seeded on decellularized bovine pericardium. In vitro, bovine pericardium samples with cell seeding were inspected on day 7, 14, and 21 by histology, scanning electron microscopy, and immunohistochemistry. In vivo, experiments were performed in nude mice by bilateral dorsal incision for the implantation of decellularized bovine pericardium with and without cell seeding. Results demonstrated that a total of 8-10 × 10(6) cells were obtained within 4-5 wk by the primary co-culture, which were detected positive for von Willebrand factor, α-smooth muscle actin antibodies, and fibronectin, indicating the presence of endothelial cells, smooth muscle cells, and fibroblasts, respectively. In vitro, the seeded cells showed a steady increase of endothelial activity from day 1 to day 7 and remained stable until day 21. After 30 days of implantation in vivo, the cells on the decellularized bovine pericardium could differentiate directionally and show all the identities of human endothelial cells, smooth muscle cells, and fibroblasts. These results indicate that the human vascular cells from the saphenous vein are an optional cell source for seeding on decellularized bovine pericardium scaffold for constructing TEHV.