Antibody-Based Vascular Targeting: Proteomic Techniques for the Identification and Quantification of Membrane Proteins on Endothelial Cells

2004 ◽  
pp. 17-38
Author(s):  
Simone Scheurer ◽  
Jascha-Nikolai Rybak ◽  
Christoph Roesli ◽  
Giuliano Elia ◽  
Dario Neri
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Vascular Targeting ◽  
Proteomic Techniques ◽  
Identification And Quantification

scholarly journals Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

2016 ◽  
Vol 124 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Zhenjun Zhao ◽  
Michael S. Johnson ◽  
Biyi Chen ◽  
Michael Grace ◽  
Jaysree Ukath ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Ionizing Radiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Vascular Targeting ◽  
Radiation Doses ◽  
Radiation Induced ◽  
Polarity Sensitive

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

Determination of Circulating Endothelial Cells and Endothelial Progenitor Cells Using Multicolor Flow Cytometry in Patients with Thrombophilia

2019 ◽  
Vol 142 (2) ◽  
pp. 113-119
Author(s):  
Martin Řádek ◽  
Eva Babuňková ◽  
Martin Špaček ◽  
Tomáš Kvasnička ◽  
Jan Kvasnička
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Endothelial Damage ◽  
Circulating Endothelial Cells ◽  
High Dose ◽  
Multicolor Flow Cytometry ◽  
Endothelial Progenitor ◽  
Identification And Quantification

Background/Aims: Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) have been described as markers of endothelial damage and dysfunction in several diseases, including deep venous thrombosis. Their role in patients with known thrombophilia has not yet been evaluated. Both EPCs and CECs represent extremely rare cell populations. Therefore, it is essential to use standardized methods for their identification and quantification. Methods: In this study, we used multicolor flow cytometry to analyze the number of EPCs and CECs in patients with thrombophilia with or without a history of thrombosis. Patients with hematological malignancies after high-dose chemotherapy and patients with acute myocardial infarction were used as positive controls. Results: EPC and CEC immunophenotypes were determined as CD45dim/–CD34+CD146+CD133+ and CD45dim/–CD34+CD146+CD133–, respectively. Increased levels of endothelial cells were observed in positive control groups. No significant changes in the number of EPCs or CECs were detected in patients with thrombophilia compared to healthy controls. Conclusion: Our optimized multicolor flow cytometry method allows unambiguous identification and quantification of endothelial cells in the peripheral blood. Our results support previous studies showing that elevated levels of CECs could serve as an indicator of endothelial injury or dysfunction. Normal levels of CECs or EPCs were found in patients with thrombophilia.

Rab5a-mediated localization of claudin-1 is regulated by proteasomes in endothelial cells

2011 ◽  
Vol 300 (1) ◽  
pp. C87-C96 ◽  
Author(s):  
Machiko Asaka ◽  
Tetsuaki Hirase ◽  
Aiko Hashimoto-Komatsu ◽  
Koichi Node
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Tight Junction ◽  
Endothelial Permeability ◽  
Ubiquitin Proteasome System ◽  
Cell Contact ◽  
Major Barrier ◽  
Tight Junction Permeability ◽  
Interfering Rna

Tight junctions composed of transmembrane proteins, including claudin, occludin, and tricellulin, and peripheral membrane proteins are a major barrier to endothelial permeability, whereas the role of claudin in the regulation of tight junction permeability in nonneural endothelial cells is unclear. This study demonstrates that claudin-1 is dominantly expressed and depletion of claudin-1 using small interfering RNA (siRNA) increased tight junction permeability in EA hy.926 cells, indicating that claudin-1 is a crucial regulator of endothelial tight junction permeability. The ubiquitin-proteasome system has been implicated in the regulation of endocytotic trafficking of plasma membrane proteins. Therefore, the involvement of proteasomes in the localization of claudin-1 was investigated by pharmacological and genetic inhibition of proteasomes using a proteasome inhibitor, N-acetyl-Leu-Leu-Nle-CHO, and siRNA against the β5-subunit of the 20S proteasome, respectively. Claudin-1 was localized at cell-cell contact sites in control cells. Claudin-1 was localized in the cytoplasm in association with Rab5a and EEA-1, a marker of early endosome, following inhibition of proteasomes. Depletion of Rab5a using siRNA reversed the localization of claudin-1 induced by inhibition of proteasomes. These data suggest that proteasomes regulate claudin-1 localization at the plasma membrane, which changes upon proteasomal inhibition to a Rab5a-mediated endosomal localization.

Vascular Disrupting Combretastatins Impair Bone Marrow Endothelial Cells by Depolymerizing the Microtubule Cytoskeleton.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2183-2183
Author(s):  
Raphael C Bosse ◽  
Sunil Rohatgi ◽  
Amy Meacham ◽  
Elizabeth Wise ◽  
Edward W Scott ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Research Funding ◽  
Capillary Tube ◽  
Phase 1 ◽  
Vascular Targeting ◽  
Microtubule Cytoskeleton ◽  
Xtt Assay ◽  
Scratch Assay ◽  
Vascular Disrupting

Abstract Abstract 2183 Background: The vascular disrupting combretastatins CA1P (OXi4503) and CA4P (Zybrestat) are known to target solid tumor neovasculature by altering endothelial cell morphology and occluding cancer neovessels. In prior work, we and others have demonstrated that combretastatins target acute myeloid leukemia (AML) cells by generating reactive oxygen species within the malignant myeloid cells. We are currently conducting a phase 1 clinical trial of OXi4503 in patients with AML (NCT01085656). However, the vascular targeting mechanisms by which combretastatins regress AML are ill defined. We hypothesized that combretastatins also target bone marrow endothelial cells (BMECs). Methods: BMECs were isolated and cultured from bone marrow of healthy subjects and patients with AML. The cells were treated with various doses of the combretastatins CA1 and CA4 for 24 and 48 hours. Cell viability was measured by XTT assay. BMEC function in response to combretastatin treatments was quantified using a modified in vitro scratch assay and 2D capillary formation using Matrigel. Analysis of microtubule cytoskeleton was performed by staining treated BMECs with anti-β-tubulin antibodies and fluorescence microscopy. To ascertain the effects of combretastatin treatment on the ability of BMECs to support and protect AML cells, co-culture experiments were performed. Results: The combretastatins CA1 and CA4 did not decrease BMEC viability or metabolic activity. However, scratch assay showed impaired BMEC migration, with only 15.6% and 7.5% of the wound covered by BMECs after CA1 and CA4 respectively, compared to untreated control (80%) (p=0.0173). Matrigel assay also showed impaired BMEC function after combretastatin treatment, with complete lack of capillary tube formation compared to untreated BMECs which generated an average of 27.3 capillary tubes per 15 BMECs (p<0.001). Immunohistochemistry showed a distinct degradation of microtubule cytoskeleton in BMECs treated with CA1 and CA4. Finally, when treated with a vascular targeting combretastatin, AML cells co-cultured with BMECs showed a lower survival rate than AML cells alone (64.1% vs. 75.3%, p = 0.0267). Conclusion: Combretastatins directly impair BMEC function and eliminate BMEC protection of AML cells. The dual targeting of combretastatins on BMECs and AML cells leads to enhanced AML regression. These data highlight the role of the leukemia microenvironment as a protective reservoir of disease and provide methods of tracking biologic activity of combretastatins in early phase clinical studies. Disclosures: Cogle: OXiGENE: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding.

BIOSYNTHESIS AND EXPRESSION OF FACTOR V IN MAGAKARYOCYTES

1987 ◽  
Author(s):  
R W Colman ◽  
A Gewirtz ◽  
D L Wang ◽  
M M Huh ◽  
B P Schick ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Guinea Pig ◽  
Light Chain ◽  
Critical Role ◽  
Small Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor V ◽  
Single Chain ◽  
Activation Peptide

Coagulation factor V (FV), is a single chain, multifunctional glycoprotein of Mr 350,000 which interacts with a variety of hemostatic proteins such as factor Xa, prothrombin, thrombin and protein C, on the surface of platelets and vascular endothelial cells. FV serves as both a cofactor and substrate in the generation of thrombin and plays a critical regulatory role in both physiologic hemostasis and pathologic thrombosis. The biosynthesis of FV and its subsequent expression are therefore expected to be precisely controlled and may differ in the three sites of synthesis - hepatocytes, endothelial cells, and megakaryocytes (MK). We have previously demonstrated that each guinea pig MK contains 500 times as much FV as in a platelet, as quantified by a competitive enzyme-linked-immunosorbent assay and expresses FV by cytoimmunofluorescence. De novo biosynthesis was demonstrated by incorporation of S-methionine into FV purified on a immunoaffinity column. The purified MK protein exhibited both FV coagulant activity and antigenicity. However, MK FV was more slowly activated by thrombin, more stable in the absence of Ca and exhibited a slightly higher M of 380,000 compared to plasma FV. Similar studies have documented biosynthesis in human MK. In addition, all morphologically recognizable MK enriched by elutriation from human bone marrow contained FV as documented by both monospecific polyclonal and monoclonal antibodies (MAb) to FV. All these cells bound FV since a murine MAb reacting with the light chain of FV (B38) labeled all cells. In contrast, 68% of cells synthesized FV since B10, a MAb to the activation peptide recognizing FV but not FVa, labeled this fraction. To determine whether immature nonnorphologically recognizable MK expressed FV, we identified these cells with an antiserum to human platelet glycoproteins and then probed them with B38. Seventy percent (70%) of such small cells expressed FV. In contrast, no small cells in MK colonies cloned in FV deficient medium expressed FV while only 40% of such colonies contained cells which expressed FV.To further probe the regulation of FV in MK we attempted to correlate the synthesis of FV as probed by MAb B10 with geometric mean cell diameter, stage and ploidy. No significant correlation of FV with any of these indicators of MK maturation. In contrast, preliminary studies suggest that low doses of tetradecanoyl phorbol acetate augment both the number of MK containing FV and the level of FV expressed by individual cells. Thus, FV synthesis may be regulated independent of size, stage, or ploidy and protein kinase C may play a role.To further define the molecular nature of FV in MK we found that purified FV was converted from a monomer to high Mr multimers by an enzyme derived from MK. These multimers resulting from covalent crosslinking since they were stable to SDS, 100° C and reducing agents. The responsible enzyme appeared to be MK FXIIIa since it required C, was inhibited by agents which react with the active site thiol group and was blocked by pseudoamine donor substrates such as putrescine. In addition, FXIIIa was directly demonstrated in guinea pig MK by a specific activity stain. Other investigators have established that FV became irreversibly associated with platelet cytoskeletons after exposure to thrombin. tested whether FXIIIa might mediate this association by performing ligand blotting of platelet membrane proteins using 125I-FV(FV*). Only actin of all the membrane proteins was detected by radioautography. The binding of FV* to the cytoskeleton was dependent in the presence of Ca and FXIIIa. In purified systems crosslinked complexes containing FV* or radiolabeled actin were detected in separate experiments. In whole platelets, the formation of the heteropolymer, after thrombin stimulation, was inhibited by antibodies to FXIII a chain, FV activation peptide (B10) or actin. Endogenous platelet FV was also dependent on FXIII for incorporation into the platelet cytoskeleton after thrombin stimulation. When thrombin-treated FV was crosslinked to actin only the activation peptide (150 kDa) was crosslinked. The light chain or heavy chain of FVa were not involved. Thus FXIIIa play an important role in the binding of FV in platelets to the cytoskeleton during activation and secretion.Further studies of FV in megakaryocytes are necessary to define the regulation of biosynthesis and the control of expression which dictate its critical role in hemostasis and thrombosis.

scholarly journals Proteome analysis in plant stress research: a review

2008 ◽  
Vol 43 (No. 1) ◽  
pp. 1-6 ◽  
Author(s):  
P. Vítámvás ◽  
K. Kosová ◽  
I.T. Prášil
Keyword(s):  
Plant Resistance ◽  
Posttranslational Modifications ◽  
Plant Stress ◽  
Proteome Analysis ◽  
Stress Research ◽  
Important Approach ◽  
Related Proteins ◽  
Proteomic Techniques ◽  
Identification And Quantification

Proteomic techniques that allow the identification and quantification of stress-related proteins, mapping of dynamics of their expression and posttranslational modifications represent an important approach in the research of plant stresses. In this review, we show an outline of proteomics methods and their applications in the research of plant resistance to various types of stresses.

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