Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.

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Dissociation between alveolar transmigration of neutrophils and lung injury in hyperoxia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00067.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. L1050-L1058 ◽

Cited By ~ 35

Author(s):

Sandra Perkowski ◽

Arnaud Scherpereel ◽

Juan-Carlos Murciano ◽

Evguenia Arguiri ◽

Charalambos C. Solomides ◽

...

Keyword(s):

Cell Adhesion ◽

Lung Injury ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Wild Type ◽

Pulmonary Endothelium ◽

Pulmonary Uptake ◽

Endothelial Surface ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1

The objective of this study was to quantitatively assess changes in cell adhesion molecule (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O2 for 0–72 h, and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAM expression was quantified with a dual-radiolabeled MAb technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking MAbs to murine P-selectin, ICAM-1, or platelet-endothelial cell adhesion molecule-1 (PECAM-1) were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. PMN sequestration occurred within 8 h of hyperoxia, although alveolar emigration occurred later (between 48 and 72 h), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1, and PECAM-1, reflecting an increase in their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking PMN recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.

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Single-Chain Variable Fragment Antibody of Vascular Cell Adhesion Molecule 1 as a Molecular Imaging Probe for Colitis Model Rabbit Investigation

Contrast Media & Molecular Imaging ◽

10.1155/2019/2783519 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8

Author(s):

Chunbao Liu ◽

Jun Zhou ◽

Xiaojie Cheng ◽

Liang Xia ◽

Junfen Zhou ◽

...

Keyword(s):

Cell Adhesion ◽

Molecular Imaging ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Vascular Cell Adhesion ◽

Vascular Cell ◽

Cell Adhesion Molecule 1 ◽

Colitis Model

Vascular cell adhesion molecule-1 (VCAM-1) can be a promising target for colitis study because of its critical role in inflammation development. Single-chain variable fragment (scFv) antibody presents fast blood clearance when served as an imaging probe. We applied the probe of 99mTc-scFv-VCAM-1 to colitis rabbit to examine its imaging performance. The colitis model rabbit was prepared, and a typical inflammatory lesion was confirmed in the colon. The probe of 99mTc-scFv-VCAM-1 was synthesized and injected into the model animal before imaging examination. Scintigraphy detected colitis lesions in both SPECT planar and SPECT/CT fused images, with higher target-to-nontarget ratios in the model group (2.71 ± 0.31) than those in the control group (1.12 ± 0.10). Biodistribution study determined tracer uptake in different organs, and autoradiography (ARG) confirmed probe accumulation in colon lesions. The uptake ratio of the model colon to the control colon was 4.71 ± 0.61 in quantitative analysis of the ARG regions of interest. Stronger VCAM-1 expression in the model colon than that in the control colon was confirmed by western blotting and immunohistochemistry. Our imaging study indicates molecular imaging with scFv-VCAM-1 as a promising way for inflammatory bowel disease diagnosis and evaluation.

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PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets

Blood ◽

10.1182/blood-2003-06-1888 ◽

2003 ◽

Vol 102 (10) ◽

pp. 3658-3664 ◽

Cited By ~ 54

Author(s):

Vipul Rathore ◽

Michelle A. Stapleton ◽

Cheryl A. Hillery ◽

Robert R. Montgomery ◽

Timothy C. Nichols ◽

...

Keyword(s):

Feedback Loop ◽

Cell Adhesion Molecule ◽

Platelet Adhesion ◽

Chain Complex ◽

Wild Type ◽

Platelet Endothelial Cell Adhesion ◽

Von Willebrand ◽

Cell Adhesion Molecule 1 ◽

Activation Motif ◽

Willebrand Factor

AbstractPlatelet adhesion at sites of vascular injury is mediated, in part, by interaction of the platelet plasma membrane glycoprotein (GP) Ib/V/IX complex with von Willebrand Factor (VWF) presented on collagen-exposed surfaces. Recent studies indicate that GPIb/V/IX may be functionally coupled with the Fc receptor γ (FcRγ)-chain, which, by virtue of its cytoplasmic immunoreceptor tyrosine-based activation motif, sends activation signals into the cell. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an inhibitory receptor that has previously been shown to negatively regulate platelet responses to collagen, which transduces activation signals via the GPVI/FcRγ-chain complex. To determine whether PECAM-1 might similarly regulate signals emanating from GPIb/FcRγ, we compared activation and aggregation responses to VWF of PECAM-1-positive and PECAM-1-deficient murine platelets. PECAM-1 and the FcRγ-chain became rapidly tyrosine phosphorylated in platelets following botrocetin-induced VWF binding, but FcRγ-chain tyrosine phosphorylation was delayed in PECAM-1-positive, versus PECAM-1-deficient, platelets. PECAM-1-deficient platelets were hyperaggregable to VWF, exhibited enhanced spreading and, under conditions of arterial flow, formed markedly larger thrombi on immobilized VWF than did wild-type platelets. Taken together, these data support the notion that engagement of the GPIb complex, in addition to sending activation signals, also initiates a negative feedback loop involving PECAM-1 that controls the rate and extent of platelet activation. (Blood. 2003;102:3658-3664)

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Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56lck

Blood ◽

10.1182/blood.v97.8.2351 ◽

2001 ◽

Vol 97 (8) ◽

pp. 2351-2357 ◽

Cited By ~ 97

Author(s):

Debra K. Newman ◽

Christin Hamilton ◽

Peter J. Newman

Keyword(s):

T Cells ◽

Endothelial Cell ◽

Cell Adhesion Molecule ◽

Antigen Receptor ◽

Inhibitory Effect ◽

Wild Type ◽

Antigen Receptor Signaling ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion

Abstract Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene superfamily that is expressed on the surface of platelets, endothelial cells, myeloid cells, and certain lymphocyte subsets. PECAM-1 has recently been shown to contain functional immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain, and co-ligation of PECAM-1 with the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and attenuation of TCR-mediated cellular signaling. To determine the molecular basis of PECAM-1 inhibitory signaling in lymphocytes, the study sought to (1) establish the importance of the PECAM-1 ITIMs for its inhibitory activity, (2) determine the relative importance of SHP-2 versus SHP-1 in mediating the inhibitory effect of PECAM-1, and (3) identify the protein tyrosine kinases required for PECAM-1 tyrosine phosphorylation in T cells. Co-ligation of wild-type PECAM-1 with the B-cell antigen receptor expressed on chicken DT40 B cells resulted in a marked reduction of calcium mobilization—similar to previous observations in T cells. In contrast, co-ligation of an ITIM-less form of PECAM-1 had no inhibitory effect. Furthermore, wild-type PECAM-1 was unable to attenuate calcium mobilization in SHP-2–deficient DT40 variants despite abundant levels of SHP-1 in these cells. Finally, PECAM-1 failed to become tyrosine phosphorylated in p56lck-deficient Jurkat T cells. Together, these data provide important insights into the molecular requirements for PECAM-1 regulation of antigen receptor signaling.

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Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions

Blood ◽

10.1182/blood.v98.5.1456 ◽

2001 ◽

Vol 98 (5) ◽

pp. 1456-1463 ◽

Cited By ~ 87

Author(s):

Karen L. Jones ◽

Sascha C. Hughan ◽

Sacha M. Dopheide ◽

Richard W. Farndale ◽

Shaun P. Jackson ◽

...

Keyword(s):

Cell Adhesion Molecule ◽

Collagen Matrix ◽

Negative Regulator ◽

Dependent Manner ◽

Wild Type ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Dose Dependent ◽

Deficient Mice ◽

Endothelial Cell Adhesion

The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin–ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and FcγRIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1–immunoglobulin chimeras and platelets derived from PECAM-1–deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)–, and PECAM-1–immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time- and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1–immunoglobulin chimera, but not mutant K89A PECAM-1–immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen- and CRP-induced platelet aggregation. PECAM-1–deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1–immunoglobulin chimera. Platelets derived from PECAM-1–deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds−1 compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.

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Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00035.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. H485-H500 ◽

Cited By ~ 35

Author(s):

Sparkle Russell-Puleri ◽

Nathaniel G. dela Paz ◽

Diana Adams ◽

Mitali Chattopadhyay ◽

Limary Cancel ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Cell Adhesion Molecule ◽

Fluid Shear Stress ◽

Wild Type ◽

Fluid Shear ◽

Platelet Endothelial Cell Adhesion ◽

Cox 2 ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2. Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5β1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1−/− mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals. NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

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Peroxynitrite reduces the endothelium-derived hyperpolarizing factor component of coronary flow-mediated dilation in PECAM-1-knockout mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00424.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. R57-R65 ◽

Cited By ~ 20

Author(s):

Yanping Liu ◽

Aaron H. Bubolz ◽

Yang Shi ◽

Peter J. Newman ◽

Debra K. Newman ◽

...

Keyword(s):

Nitric Oxide ◽

Signal Transduction ◽

Coronary Flow ◽

Cell Adhesion Molecule ◽

Coronary Circulation ◽

Flow Mediated Dilation ◽

Wild Type ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits flow-mediated dilation (FMD), we examined whether steady-state FMD in mouse coronary arteries (MCAs) is affected in the PECAM-1 knockout (KO) mouse. MCAs were isolated from wild-type (WT) or KO mice and prepared for videomicroscopy, histofluorescence, Western blotting, and immunohistochemistry. FMD was examined in the absence and presence of Nω-nitro-l-arginine methyl ester (l-NAME) and l-NAME+indomethacin (INDO). FMD was reduced in KO relative to WT MCAs, but the l-NAME-inhibitable portion of FMD was similar between the two. The INDO-sensitive component of FMD was diminished in KO MCAs. In contrast, the residual component of dilation, presumably because of endothelium-derived hyperpolarizing factor (EDHF), was abolished in KO MCAs. Histofluorescence showed relatively more superoxide (O2−·; oxy-ethidium fluorescence) and peroxide production (dihydrochlorofluorescene fluoresecence) in KO MCAs at rest. Flow augmented O2−· and peroxide production in WT MCAs but had little effect on KO MCAs. Enhanced nitric oxide generation was observed in arteries from KO mice, accompanied with increased eNOS S1177 phosphorylation. In vessels from KO mice, treatment with ebselen decreased peroxynitrite (ONOO−) formation and improved the reduced FMD, largely due to restoration of the presumed EDHF component. These results suggest that PECAM-1 is necessary for normal FMD in the mouse coronary circulation. In the absence of this adhesion and signaling molecule, ONOO− production is increased concomitant with a reduction in both the EDHF and INDO-sensitive components of FMD.

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Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza

Vaccines ◽

10.3390/vaccines8010118 ◽

2020 ◽

Vol 8 (1) ◽

pp. 118

Author(s):

Deimante Lukosaityte ◽

Jean-Remy Sadeyen ◽

Angita Shrestha ◽

Joshua E. Sealy ◽

Sushant Bhat ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Avian Influenza ◽

Influenza Viruses ◽

H9n2 Virus ◽

Control Measures ◽

Mitigation Measures ◽

Passive Immunisation ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Recombinant Scfv

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

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The immunoreactivity of the anti-p21Ras single-chain fragment variant KGH-R1 and its predicted binding sites to p21Ras

Immunotherapy ◽

10.2217/imt-2019-0222 ◽

2020 ◽

Vol 12 (12) ◽

pp. 879-890

Author(s):

Peng Wang ◽

Xinyan Pan ◽

Qiang Feng ◽

Hong Zou ◽

Jing Cui ◽

...

Keyword(s):

Binding Sites ◽

Protein Structures ◽

Antibody Fragment ◽

Human Tumor ◽

Wild Type ◽

Single Chain ◽

Single Chain Antibody ◽

Chain Fragment ◽

Complementarity Determining Regions ◽

Single Chain Fragment

Aim: Previously, we constructed a novel anti-p21Ras single-chain antibody fragment, KGH-R1-single-chain fragment variant (ScFv). However, the immunoreactivity of this antibody toward p21Ras is still unclear. Materials & methods: ELISAs, immunohistochemistry, western blotting and immunofluorescence were used to identify the immunoreactivity of KGH-R1-ScFv toward p21Ras. An in silico approach was used to determine the protein structures of KGH-R1-ScFv and p21Ras and then to predict the site involved in the binding of KGH-R1-ScFv to p21Ras. Results: KGH-R1-ScFv had a specific immune reaction with prokaryotically expressed p21Ras, human tumor cells and tumor tissues with RAS mutations or overexpression of RAS. Molecular docking showed that KGH-R1-ScFv could stably interact with wild-type and mutant p21Ras and the binding sites were in the complementarity-determining regions of KGH-R1-ScFv. Conclusion: KGH-R1-ScFv shows specific immunoreactivity toward wild-type and mutant p21Ras as well as the corresponding tumors, which suggests that KGH-R1-ScFv shows potential as a therapeutic antibody for therapy of RAS-driven tumors.

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Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

The Journal of Cell Biology ◽

10.1083/jcb.124.1.195 ◽

1994 ◽

Vol 124 (1) ◽

pp. 195-203 ◽

Cited By ~ 71

Author(s):

HM DeLisser ◽

J Chilkotowsky ◽

HC Yan ◽

ML Daise ◽

CA Buck ◽

...

Keyword(s):

Cell Adhesion ◽

Ligand Binding ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Cytoplasmic Domain ◽

Wild Type ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion ◽

Cell Cell

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild-type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.

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