Vein Endothelial Cells Research Articles

The use of magnetron sputtering for the deposition of thin titanium coatings on the surface of bioresorbable electrospun fibrous scaffolds for vascular tissue engineering: A pilot study

The deposition of thin titanium coatings using magnetron spattering on the surface of bioresorbable fibrous scaffolds produced by electrospinning was investigated. Parameters that allow the surface modification without damaging the “macro” structure of scaffolds were determined. Physicochemical properties of the modified scaffolds were described using SEM, EDS, DSC, optical goniometry, and mechanical testing. It was shown that plasma treatment has a significant influence on the scaffolds’ fiber surface relief. The modification process leads to a slight decrease of the scaffold mechanical performance mainly caused by polymer crystallization. Increasing the deposition time increases the amount of titanium on the surface. The biocompatibility of the modified scaffolds was studied using hybridoma of the endothelial cells of human umbilical vein and human lung carcinoma (EA.hy 926 cell line). Cell adhesion, viability, and secretion of interleukin-6 (IL6), interleukin-8 (IL8), and vascular endothelial growth factor (VEGF) were investigated. It was demonstrated that the deposition of thin titanium coatings on the fibrous scaffolds’ surface enhances cell adhesion. Additionally, it was determined that modified scaffolds have proangiogenic activity.

Recognition of extremophilic archaeal viruses by eukaryotic cells: a promising nanoplatform from the third domain of life.

Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration of novel medical nanoplatforms. Here, we selected two archaeal viruses Sulfolobus monocaudavirus 1 (SMV1) and Sulfolobus spindle shaped virus 2 (SSV2) owing to their unique spindle shape, hyperthermostable and acid-resistant nature and studied their interaction with mammalian cells. Accordingly, we followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path for selective cell targeting. On internalization, both viruses localize to the lysosomal compartments. Neither SMV1, nor SSV2 induced any detrimental effect on cell morphology, plasma membrane and mitochondrial functionality. This is the first study demonstrating recognition of archaeal viruses by eukaryotic cells which provides good basis for future exploration of archaeal viruses in bioengineering and development of multifunctional vectors.

Mesenchymal stem cell-conditioned media ameliorate diabetic endothelial dysfunction by improving mitochondrial bioenergetics via the Sirt1/AMPK/PGC-1α pathway

Vasculopathy is a major complication of diabetes. Impaired mitochondrial bioenergetics and biogenesis due to oxidative stress are a critical causal factor for diabetic endothelial dysfunction. Sirt1, an NAD+-dependent enzyme, is known to play an important protective role through deacetylation of many substrates involved in oxidative phosphorylation and reactive oxygen species generation. Mesenchymal stem cell-conditioned medium (MSC-CM) has emerged as a promising cell-free therapy due to the trophic actions of mesenchymal stem cell (MSC)-secreted molecules. In the present study, we investigated the therapeutic potential of MSC-CMs in diabetic endothelial dysfunction, focusing on the Sirt1 signalling pathway and the relevance to mitochondrial function. We found that high glucose-stimulated MSC-CM attenuated several glucotoxicity-induced processes, oxidative stress and apoptosis of endothelial cells of the human umbilical vein. MSC-CM perfusion in diabetic rats ameliorated compromised aortic vasodilatation and alleviated oxidative stress in aortas. We further demonstrated that these effects were dependent on improved mitochondrial function and up-regulation of Sirt1 expression. MSC-CMs activated the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), leading to direct interaction between Akt and Sirt1, and subsequently enhanced Sirt1 expression. In addition, both MSC-CM and Sirt1 activation could increase the expression of peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), as well as increase the mRNA expression of its downstream, mitochondrial, biogenesis-related genes. This indirect regulation was mediated by activation of AMP-activated protein kinase (AMPK). Overall our findings indicated that MSC-CM had protective effects on endothelial cells, with respect to glucotoxicity, by ameliorating mitochondrial dysfunction via the PI3K/Akt/Sirt1 pathway, and Sirt1 potentiated mitochondrial biogenesis, through the Sirt1/AMPK/PGC-1α pathway.

C1ql1/Ctrp14 and C1ql4/Ctrp11 promote angiogenesis of endothelial cells through activation of ERK1/2 signal pathway.

C1ql-like (C1QL)-1 and -4 proteins are encoded by homologous genes that are highly expressed in brain and adipose tissues. However, functional properties of C1QL proteins outside of the brain and adipocytes remain unknown. Here, we report that the globular domain of C1ql1/Ctrp14 and C1ql4/Ctrp11 proteins directly stimulate the angiogenesis of endothelial cells. In this study, soluble C1ql1/CTRP14 and C1ql4/Ctrp11 proteins, produced in prokaryote expression system, are co-cultured with human umbilical vein endothelium cells (HUVECs), which phenotype is identified with von Willebrand factor antibody. C1ql1/Ctrp14 and C1ql4/Ctrp11 promote the migration and capillary tube formation of HUVECs in a dose-dependent manner. During this process, phosphorylation of c-Raf, MEK1/2, ERK1/2, and p90RSK are activated by C1ql1/Ctrp14 and C1ql4/Ctrp11. MEK1/2 inhibitor, U0126, blocks C1ql1/Ctrp14-, and C1ql4/Ctrp11-induced capillary tube formation and cell migration. Moreover, the immunoreactivity of the receptor of C1QL1-C1QL4, brain-specific angiogenesis inhibitor 3 (BAI3), is detected in HUVECs, suggesting that BAI3 may mediate C1QL1/CTRP14- and C1QL4/CTRP11-induced angiogenesis. Meanwhile, C1ql1/Ctrp14 and C1ql4/Ctrp11 exposure also causes a stimulatory response of angiogenesis in chick yolk sac membrane. These data demonstrate that C1ql1/Ctrp14 and C1ql4/Ctrp11 stimulate the new blood vessel growth by activation of ERK1/2 signal pathway. The proangiogenic activity of C1ql1/Ctrp14 and C1ql4/Ctrp11 provides novel insights into the new opportunities for therapeutic intervention by targeting C1QLs in tumorigenesis, tissue regeneration, and recovery of ischemic heart disease.

The Diagnostic Impact of C4d, CD68, and NF-κB Expression in the Differentiation Between Recurrent Hepatitis C and Acute Cellular Rejection After Liver Transplantation.

Liver transplantation is the selected treatment for patients with advanced liver disease and cirrhosis, mostly as a complication of hepatitis C virus (HCV). Recurrent HCV and acute cellular rejection (ACR) of the graft are the most common causes of graft failure. The distinction between the 2 conditions is essential because they are managed differently. In some cases, the clinical and histopathologic features may overlap between recurrent hepatitis C and ACR, making differentiation difficult. The aim of this study was to investigate the role of C4d, CD68, and nuclear factor kappa-B (NF-κB) in the differentiation between ACR and recurrent HCV in the post-liver-transplant biopsy using immunohistochemistry. C4d expression in endothelial cells of portal or central veins (P=0.001) and the number of macrophages highlighted by CD68 (P=0.02) were in favor of ACR, whereas NF-κB expression by hepatocytes was in favor of recurrent hepatitis C. Vascular injury demonstrated by endothelial expression of C4d and prominent macrophage infiltration identified by CD68 expression were the distinguishing criteria for ACR and representing humoral and cellular-mediated immunity as evoking factors for graft injury. The upregulation of NF-κB in the hepatocytes of recurrent hepatitis C could be an immune response to infection or it may be induced by HCV itself.

P17 Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Endothelial cells are routinely exposed to elevated glucose concentrations post-prandially in healthy individuals and permanently in patients with metabolic syndrome and diabetes. To address the role of endothelium in glucose uptake and supply to underlying tissues in these conditions, we assessed the presence of glucose transporters (GLUTs) in endothelial cells and their sugar transport capabilities in response to high glucose. In human umbilical vein (HUVEC), saphenous vein, microdermal vessels and aortic endothelial cells, GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT6 (SLC2A6), and in microdermal vessels also GLUT12 (SLC2A12), were the main glucose transporters as assessed by mRNA, with no fructose transporters nor sodium-dependent glucose transporter 1 (SLC5A1) detected. Uptake of 14C-fructose was negligible. GLUT1 and GLUT3 proteins were detected in all cell types and were responsible for ~60% glucose uptake in HUVECs, where both GLUT1 and GLUT3, but not GLUT6 siRNA knock-down, reduced the transport. Under shear conditions, GLUT1 protein decreased, GLUT3 increased, and 14C-deoxy-glucose uptake was attenuated. In high glucose, lipid storage was increased, cell numbers were lower, 14C-deoxy-glucose uptake decreased owing to attenuated GLUT3 protein and less surface GLUT1, and trans-endothelial transport of glucose increased due to cell layer permeability changes. We conclude that glucose transport by endothelial cells is relatively resistant to effects of elevated glucose. Cells would continue to supply it to the underlying tissues at a rate proportional to the blood glucose concentration, independent of insulin or fructose.

Abstract 3042: The novel Akt inhibitor AZD5363 following radiotherapy improves tumor control in mouse models of head and neck cancer

Abstract Radiation therapy (RT) is an important anti-cancer treatment. Understanding the molecular pathways involved in the response of tumors to RT has led to interest in developing drugs which target these; one such target is Akt. Previous studies support the hypothesis that Akt inhibition might enhance the radiosensitivity of tumors through modification of cellular and micro-environmental factors. AZD5363 is a novel, orally bio-available, ATP competitive inhibitor of Akt and here we have evaluated whether AZD5363 will increase the efficacy of RT in preclinical models of head and neck (H&N) cancer. The ability of AZD5363 to inhibit the Akt pathway was investigated in vitro in 5 H&N cell lines and in vivo (FaDu xenografts) using western blot and ELISA techniques. The effect of AZD5363 on the radiosensitivity in vitro was assessed using clonogenic assays. The FaDu xenograft model was used to test the effect of AZD5363 on tumor growth when used as a sequential treatment (for 7 days before RT) or adjuvant therapy (continuously after RT). RT was given as a 6Gy single dose. The effect of these treatments on the tumor microenvironment prior to and after irradiation was also assessed using FAZA PET/CT, immunohistochemistry and ELISA techniques. AZD5363 effectively inhibited the Akt pathway in in vitro and in vivo. There was no effect on radiosensitivity in vitro when drug was administered in a variety of sequences and durations around RT. In vivo, there was no improvement in tumor control when AZD5363 was given for 7 days before RT. But survival was improved (p = 0.006) when AZD5363 was administered continuously after RT with reduced tumor growth (p = 0.0035, 7 days after RT). There was no effect of drug alone on tumor growth and no difference in the level of tumor hypoxia (measured using FAZA PET/CT or pimonidazole binding) after 7 days AZD5363. Human VEGF and HIF1α levels were reduced (p = 0.0104 and p = 0.002, respectively) after 7 days AZD5363. There was no difference in microvessel density (MVD) prior to RT, but 7 days after, MVD was significantly reduced if adjuvant AZD5363 had been given (p = 0.0317). AZD5363 was also found to effect the rate of proliferation of human umbilical vein endothelial (HUVEC) cells in vitro. AZD5363 effectively improves long term control of FaDu tumors when combined with RT. However, this only occurs when the drug is used in the adjuvant setting. AZD5363 has no effect on the radiosensitivity of H&N cells in vitro, which suggests that the in vivo effects are dependent on the tumor microenvironment. AZD5363 reduces tumor VEGF and HIF1α in an oxygen independent manner and inhibits the rate of proliferation of vascular endothelial cells, effects that would reduce vascular recovery after RT. This is the first time a specific inhibitor of Akt has been reported to have this sequence dependent ability to enhance the effects of RT and has implications for how AZD5363 and potentially other Akt inhibitors are used in combination with RT in the clinic. Citation Format: Emma J. Searle, Brian Telfer, Duncan Forster, Kaye J. Williams, Barry Davies, Timothy Illidge, Ian Stratford. The novel Akt inhibitor AZD5363 following radiotherapy improves tumor control in mouse models of head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3042.

Abstract 5262: Chemopreventive and angiopreventive activity of a purified polyphenol-rich extract from olive mill wastewaters

Abstract The presence of diverse phenolic molecules in dietary elements, has been shown to prevent the occurrence of a variety of chronic and inflammatory diseases. Olives and extra virgin oil, a basic component of mediterranean diet represent an important source of these poliphenols, including hydroxytyrosol. While the strong antioxidant potential of these molecules has extensively investigated, their anti-angiogenic and chemopreventive activities remain unknown. Here we assessed the anti-angiogenic and chemopreventive activities exerted by an extracts from olive mill wastewaters (OMWWs), termed A009, which represent a waste product from olive oil industry, in vitro and in vivo on endothelial and different tumour cell lines, as compared with hydroxytyrosol alone The ability of A009 to affect cell proliferation and survival was evaluated on human umbelical endothelial vein cells (HUVECs), six different tumor cell lines, MCF-7 and MDA-MB-231 (breast), HT-29 and HCT-116 (colon) PC-3 and DU-145 (prostate) and the murine CT26 CRC cells by MTT assay, while the induction of apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry. Functional studies evaluated the capacity of OMWWs to interfere with endothelial cell tube formation, migration and invasion by morphogenesis and Boyden chamber assays, respectively. Finally, the inhibition of angiogenesis and tumor cell growth was evaluated in vivo, by the matrigel sponge assay and tumor xenograft. The A009 extract was able to inhibit both HUVECs and tumor cell lines growth in a dose dependent manner, exerting a stronger inhibitory effect as compared to the pure hydroxytyrosol alone. This effect was directly associated with the induction of apoptosis and ROS on HUVECs. HT-29 anc HCT-116 CRC cell lines exposed to A009 decreased thei ability to release VEGF and IL-8. Functional studies of HUVECs exposed to A009 demostrated impaired migratory and invasive abilities as far as reduced capability to form capillary-lile-structure in a dose dependent manner. Finally, A009s interfered with angiogenesis and CT26 tumor cell growth in vivo. Our results suggest that the polyphenol enriched extracts from olive oil processing (OMWWs) A009, show promising angio-preventive and chemo-preventive potentials. In particular, our data demonstrate that a pool of specific polyphenols are characterized by stronger anti-angiogenic/anti-tumor properties compared to hydroxytyrosol alone, a well known polyphenol with anti-tumor activity. Citation Format: Adriana Albini, Antonino Bruno, Barbara Bassani, Nicoletta Macrì, Francesca Caudano, Paola Corradino, Teresa Rossi, Douglas M. Noonan. Chemopreventive and angiopreventive activity of a purified polyphenol-rich extract from olive mill wastewaters. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5262.

Abstract 3244: Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer

Abstract Tumor infiltrating immune cells often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity, primarily involved in immunosurvelliance against tumors through their cytotoxic activity. We previously reported that NKs from Non Small Cell Lung Cancer (NSCLC) show the decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNγlow phenotype [Bruno et al, Neoplasia, 2014; Bruno et al, JNCI, 2014]. Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NK cells undergo an angiogenic-switch in colorectal cancer (CRC). NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling, by antibody membrane array or angiogenesis functional assay on human umbilical endothelial vein cells (HUVECs) We found that CD56brightCD16− NK cells predominate in CRC adjacent and tumor tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Further, TINK/TANKs from CRC patients express the decidual NK markers CD9 and CD49a. Secretomic analysis on CRC peripheral blood NKs revealed up regulation for several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Media conditioned by FACS sorted NK cells from peripheral blood and tumor tissue of CRC patients were able to induce HUVEC proliferation, migration and the formation of capillary like structures. Our data demonstrate that TINK/TANKS from CRC patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. We propose that TINK/TANKs could represent the hallmark for a new paradigm in CRC inflammation. 1. Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neopla sia. 2013 Feb;15(2):133-42. 2. Bruno A, Ferlazzo G, Albini A, Noonan DM. A think tank of TINK/TANKs: tumor infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis. J Natl Cancer Inst. 2014 Aug;106(8):dju200 Citation Format: Antonino Bruno, Barbara Bassani, Davide Giuseppe D’Urso, Silvia Zanellato, Elisabetta Gini, Elisa Cassinotti, Luigi Boni, Lorenzo Mortara, Adriana Albini, Douglas M. Noonan. Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3244.

SU‐G‐TeP3‐05: In Vitro Demonstration of Endothelial Dose Enhancement Due to Gold Nanoparticles During Low‐Voltage Radiotherapy

Purpose:Oraya Therapy uses low‐voltage, stereotactic, highly targeted X‐rays for the treatment of wet age‐related macular degeneration (AMD) — offering a new option for patients worldwide. Neovascular endothelial cells play a crucial role in the pathogenesis of this disease. This in‐vitro study investigates the potential of gold nanoparticles (GNP) to enhance endothelial cell damage during low‐voltage radiotherapy towards potential applications in the treatment of wet‐AMD.Methods:Primary human umbilical cord vein endothelium cells (HUVEC) were treated with 1.4 nm sized GNPs for 24 hrs and then irradiated with variable X‐ray doses using an Oraya therapy system (100 kVp) or a Small Animal Radiation and Research platform (SARRP) at other beam qualities (up to 220 kVp). Radio‐sensitization was assessed by clonogenic assays. Variable concentrations of GNPs (0.05 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml) where employed. The dose enhancement factor (DEF) was calculated as the ratio of radiation doses required to give the same biological effect (survival factor, SF) with and without GNPs.Results:Preliminary results show DEFs of up to 2.62 for the different combinations of x‐ray doses and GNP concentrations and beam qualities. In general the DEF increased with increase in GNP concentration. However, for high doses the effect of GNP becomes less apparent likely due to already high cell kill by the radiation alone.Conclusion:The findings suggest that targeted GNPs can play a significant synergistic role in enhancing stereotactic radiosurgery for wet AMD. The results also provide impetus for ongoing studies to find the optimal synergy between the doses or beam energies and GNPs concentration. This will benefit in‐vivo studies towards development of nanoparticle‐aided radiotherapy for treatment of wet‐AMD and potentially ocular cancers.

The protein PprI provides protection against radiation injury in human and mouse cells.

Severe acute radiation injuries are both very lethal and exceptionally difficult to treat. Though the radioresistant bacterium D. radiodurans was first characterized in 1956, genes and proteins key to its radioprotection have not yet to be applied in radiation injury therapy for humans. In this work, we express the D. radiodurans protein PprI in Pichia pastoris yeast cells transfected with the designed vector plasmid pHBM905A-pprI. We then treat human umbilical endothelial vein cells and BALB/c mouse cells with the yeast-derived PprI and elucidate the radioprotective effects the protein provides upon gamma irradiation. We see that PprI significantly increases the survival rate, antioxidant viability, and DNA-repair capacity in irradiated cells and decreases concomitant apoptosis rates and counts of damage-indicative γH2AX foci. Furthermore, we find that PprI reduces mortality and enhances bone marrow cell clone formation and white blood cell and platelet counts in irradiated mice. PprI also seems to alleviate pathological injuries to multiple organs and improve antioxidant viability in some tissues. Our results thus suggest that PprI has crucial radioprotective effects on irradiated human and mouse cells.

Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells

To observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs). VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

The roadmap of WT1 protein expression in the human fetal heart

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Abstract A31: Cyclic NGR peptide functionalized PEG-PLGA nanoparticles as dual-targeting carrier for site specific antitumor drug delivery

Abstract CD13 receptors are abundantly overexpressed in tumor cells as well as in neovasculature. The polymeric nanoparticles (NPs) as the nanomodules for anticancer drug carriers are well known. Nowadays, targeted delivery options are becoming a fascinating approach, so CD13 receptors was selected as a targeted site and NPs as a delivery system. By combining all the issue, a cyclic NGR (cNGR) peptide as a ligand was coupled on the terminal end of polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) and prepared the dual targeted polymeric NPs (cNGR-PEG-PTX-NPs) in order to enhance the intracellular delivery of anticancer drug to the tumor cells and tumor endothelial cells via ligand-receptor interaction. In-vitro cytotoxicity studies confirmed that the presence of cNGR enhanced the cytotoxic efficiency by 2.8 folds in human umbilical vein endothelial (HUVEC) cells, while cytotoxicity was improved by 2.6 folds in human fibrosarcoma (HT-1080) cells as compared to non-specific stealth NPs. Compared with other tested NPs, cNGR-PEG-PTX-NPs revealed more cytotoxicity by inducing more apoptosis, higher cell uptake and more G2/M phase arrest. The tumor volume inhibition rate was 59.7% in case of cNGR-PEG-PTX-NPs that was comparatively more with other formulations, indicating that cNGR-PEG-PTX-NPs could more effectively inhibit tumor growth. As a consequence, the cNGR-PEG-PTX-NPs play a key role in enhancing tumor therapeutic efficiency for treatment of CD13 receptor specific solid tumor. Citation Format: Dr. Madhu Gupta, Sr., Dr. Vikas Sharma, Prof. G.P. Agrawal, Prof. S.P. Vyas. Cyclic NGR peptide functionalized PEG-PLGA nanoparticles as dual-targeting carrier for site specific antitumor drug delivery. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A31.

Localization of Vascular Endothelial Growth Factor and Fibroblast Growth Factor 2 in the Ovary of the Ostrich (Struthio camelus).

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) play a paramount role in the regulation of normal and pathologic angiogenesis in the ovary of mammals. Very little is known on the expression of these two growth factors in the avian ovary. The aim of this study was to determine for the first time the localization of VEGF and FGF-2 in the ovary of the ostrich using immunohistochemical techniques to investigate the vascularization of the rapidly growing huge ostrich oocyte. At the oocyte periphery, distinct VEGF-positive granules are visible. In our opinion, the expression of VEGF in the growing oocytes, which does not occur in mammals such as bovines, does not significantly contribute to angiogenesis in the theca interna and externa, where all the original and developing vessels are located, but may contribute to the mitoses and survival of granulosa cells during folliculogenesis. A different immunostaining can be demonstrated for FGF-2: from late pre-vitellogenic follicles, FGF-2 immunopositivity can be observed at the inner perivitelline layer area. In the stroma, the smooth muscle cells of small arteries and the endothelial cells of venules and veins are positively stained for FGF-2. Another interesting finding of this study is the occurrence of a significant number of VEGF- and FGF-2 positive heterophilic granulocytes within the ovarian stroma, which migrate from the periphery of the ovary towards the growing follicles. We assume that the growth factors of the heterophilic granulocytes contribute significantly to the angiogenesis seen in both theca layers.

Neuroprotective effects of Potentilla fulgens on spinal cord injury in rats: an immunohistochemical analysis.

This examination was performed to research the advantage of the antioxidant impact of Potentilla fulgens on spinal cord injury (SCI) in rats. In the SCI model of this examination, the tolerably serious lesion was performed at the L1-L2 spinal segmental level. SCI animals were given P. fulgens 400 mg/kg/day, intraperitoneally. At 7 days post-lesion, exploratory rats were executed after intraperitoneal administration 7 ketamine HCL (0.15 mL/100 g body weight). Spinal cord specimens were taken for histological examination or assurance of malondialdehyde (MDA) and glutathione (GSH) levels and myelope- roxidase (MPO) action. SCI caused a remarkable decline in spinal cord GSH content, trailed by noteworthy increments in MDA levels and MPO action. Degenerative changes in some multipolar and bipolar nerve cells and pyknotic changes in the nuclei of glial cells were likewise noticed. Remarkable development was seen in cells and vascular structures of P. fulgens treated groups when contrasted with untreated groups. Potentilla fulgens application may influence angiogenetic impro- vement in vein endothelial cells, reduce inflammatory cell aggregation by influ- encing cytokine system and may make apoptotic nerve cells and neuroprotective component in glial cells.

Identification of immediate early response protein 2 as a regulator of angiogenesis through the modulation of endothelial cell motility and adhesion.

Human immediate early response2 (IER2) has been characterized as a putative nuclear protein that functions as a transcription factor or transcriptional co‑activator in the regulation of cellular responses, and may be involved in the regulation of tumor progression and metastasis. Data from our previous gene expression profile of the human microvascular endothelial cells during capillary morphogenesis showed a significant alteration of IER2 expression, suggesting that IER2 may participate in the regulation of the endothelial cell morphogenesis and angiogenesis. The aim of the present study was to investigate the role of IER2 in cell motility, cell‑matrix adhesion and invitro capillary‑like structures formation of the human umbilical vein endothelium cells (HUVECs). IER2 was constitutively expressed in HUVECs, and lentiviral‑mediated depletion of IER2 significantly reduced the cell motility, cell‑matrix adhesion and capillary‑like structures formation of HUVECs. Results also showed that depletion and overexpression of IER2 altered the actin cytoskeleton rearrangement in HUVECs. Furthermore, results from western blot analysis showed that the activity of the focal adhesion kinase (FAK) can be regulated by IER2. These results indicated that IER2 regulates endothelial cell motility, adhesion on collagen typeI matrix and the capillary tube formation, as the result of the regulation of the actin cytoskeleton rearrangement presumably via a FAK‑dependent mechanism.

Abstract 2630: Inhibition of angiogenesis contributes to enhanced radiation response in tumors following MDM2 inhibition by AMG 232

Abstract MDM2-p53 interaction and downstream signaling impacts cellular response to DNA damage. AMG 232 is a potent small molecule inhibitor that blocks the interaction of MDM2 and p53. We previously elucidated the capacity of AMG 232 to augment radiation response across a spectrum of human tumor cell lines and xenografts. In the current study, we examine the capacity of AMG 232 to inhibit angiogenesis and assess the resultant impact on AMG 232-induced radiosensitivity. We first examined the impact of AMG 232 on the growth and radiosensitivity of human umbilical vein endothelial (HUVEC) and dermal lymphatic endothelial cells (HDLEC). We found that AMG 232 inhibited the proliferation of both endothelial cell types in a dose dependent manner. Importantly, treatment with AMG 232 significantly reduced angiogenesis-related tube formation in both endothelial cell types. Using clonogenic survival analysis, we identified that treatment with AMG 232 significantly enhanced radiosensitivity. Immunoblot analysis showed that combined treatment with AMG 232 and radiation resulted in a profound impact on molecules involved in cell cycle progression, DNA damage repair, and apoptosis signaling, including FoxM1, ATM/ATR, CHK2, Rad51, and Bax. Flow cytometric analysis also showed a significant cell cycle arrest in G2/M phase with an increase of DNA damage related γH2AX level at 48 hrs following radiation and AMG 232 treatment. In H460 and SJSA-1 tumor xenografts, we found a markedly increased antitumor capacity of AMG 232 when combined with radiation. Immunohistochemical analysis revealed a significant reduction of cellular proliferation and vascular density in both tumor types following combined treatment of AMG 232 and radiation. Taken together, these findings reveal the potential anti-angiogenic capacity of AMG 232 that may contribute to augmentation of radiation response previously observed in tumor xenograft studies. A detailed investigation is in progress to examine changes in the tumor microenvironment following AMG 232 and radiation treatment, including hypoxia and vascular normalization, to assess their respective impact on tumor response to radiation. Citation Format: Lauryn R. Werner, Shyhmin Huang, Eric A. Armstrong, Fang Ma, Jude Canon, Paul M. Harari. Inhibition of angiogenesis contributes to enhanced radiation response in tumors following MDM2 inhibition by AMG 232. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2015-2630

Changes in Ca2+ Signaling and Nitric Oxide Output by Human Umbilical Vein Endothelium in Diabetic and Gestational Diabetic Pregnancies.

Diabetes (DM) complicates 3%-10% of pregnancies, resulting in significant maternal and neonatal morbidity and mortality. DM pregnancies are also associated with vascular dysfunction, including blunted nitric oxide (NO) output, but it remains unclear why. Herein we examine changes in endothelial NO production and its relationship to Ca(2+) signaling in endothelial cells of intact umbilical veins from control versus gestational diabetic (GDM) or preexisting diabetic subjects. We have previously reported that endothelial cells of intact vessels show sustained Ca(2+) bursting in response to ATP, and these bursts drive prolonged NO production. Herein we show that in both GDM and DM pregnancies, the incidence of Ca(2+) bursts remains similar, but there is a reduction in overall sustained phase Ca(2+) mobilization and a reduction in NO output. Further studies show damage has occurred at the level of NOS3 protein itself. Since exposure to DM serum is known to impair normal human umbilical vein endothelial cell (HUVEC) function, we further studied the ability of HUVEC to signal through Ca(2+) after they were isolated from DM and GDM subjects and maintained in culture for several days. These HUVEC showed differences in the rate of Ca(2+) bursting, with DM > GDM = control HUVEC. Both GDM- and DM-derived HUVEC showed smaller Ca(2+) bursts that were less capable of NOS3 activation compared to control HUVEC. We conclude that HUVEC from DM and GDM subjects are reprogrammed such that the Ca(2+) bursting peak shape and duration are permanently impaired. This may explain why ROS therapy alone is not effective in DM and GDM subjects.

Influence of Jagged1 on apoptosis-related gene expression: a microarray database analysis

Notch signaling regulates various cell behaviors including cell death and survival. The present study aimed to investigate the influence of Notch ligand, Jagged1, on the expression of apoptosis related genes using the microarray database analysis. In this regard, microarray dataset from Gene Expression Omnibus (GEO) repository was searched with keywords. The included GEO series were downloaded and analyzed using the Connection Up- and Down-Regulation Expression Analysis of Microarrays Extension program. The predicted protein–protein interaction was determined using GenesFANs program. The results demonstrated that Jagged1 enhanced pro-apoptosis gene expression in human lung and breast cancer cell line while promoted the expression of anti-apoptosis gene in human lymphatic epithelial cells. In human umbilical endothelial vein cells, Jagged1 promoted both pro- and anti-apoptosis gene expressions. No significant change in apoptotic related gene was noted in Jagged1 overexpressing human endometrial stromal cells. However, Jagged1 down-regulated both pro- and anti-apoptosis gene in human lung and breast cancer cell line as well as human umbilical endothelial vein cells. In conclusion, Jagged1 regulated both pro- and anti-apoptosis gene expressions, depending on cell types. However, the influence of Jagged1 on cell apoptosis or survival is needed further confirmation experiments.