Vascular endothelial effects of collaborative binding to platelet/endothelial cell adhesion molecule-1 (PECAM-1).

Targeting drugs to endothelial cells has shown the ability to improve outcomes in animal models of inflammatory, ischemic and thrombotic diseases. Previous studies have revealed that certain pairs of ligands (antibodies and antibody fragments) specific for adjacent, but distinct, epitopes on PECAM-1 enhance each other's binding, a phenomenon dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL. This discovery has been leveraged to enable simultaneous delivery of multiple therapeutics to the vascular endothelium. Given the known role of PECAM-1 in promoting endothelial quiescence and cell junction integrity, we sought here to determine if CEPAL might induce unintended vascular effects. Using a combination of in vitro and in vivo techniques and employing human and mouse endothelial cells under physiologic and pathologic conditions, we found only modest or non-significant effects in response to antibodies to PECAM-1, whether given solo or in pairs. In contrast, these methods detected significant elevation of endothelial permeability, pro-inflammatory vascular activation, and systemic cytokine release following antibody binding to the related endothelial junction protein, VE-Cadherin. These studies support the notion that PECAM-1-targeted CEPAL provides relatively well-tolerated endothelial drug delivery. Additionally, the analysis herein creates a template to evaluate potential toxicities of vascular-targeted nanoparticles and protein therapeutics.

Recurrent infections and immunological dysfunction in congenital disorder of glycosylation Ia (CDG Ia).

Congenital disorder of glycosylation Ia is the most common defect of glycosylation and is due to mutations in phosphomannomutase 2. This leads to aberrant N-linked oligosaccharides. The phenotype of CDG Ia reflects the essential nature of glycosylation and patients typically present with multiple organs affected, with hypotonia, developmental delay, inverted nipples and abnormal fat pads. Later features include retinitis pigmentosa, stroke, cerebellar atrophy and malabsorption. Approximately 20% of patients die in the first year of life and infection is the most common cause of death. Immunological function has not previously been investigated in these patients and the critical role of oligosaccharides on adhesion molecules suggested that haematopoietic cell migration and communication could be disrupted by mutations in phosphomannomutase 2. We characterized the clinical features, performed standard immunological evaluations, and performed specific analyses of neutrophil adhesion molecules on two patients to address this question. Patient neutrophils had diminished chemotaxis but expressed comparable levels of adhesion molecules and rolled on artificial endothelium equivalently to control neutrophils. The most significant feature of the patients' immunological function was poor vaccine responses. These two affected patients were begun on intravenous immunoglobulin with some improvement in their infections.

Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis.

Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitro studies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivo angiogenesis. Both receptors were detected on vascular endothelium in situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels.

A role for hyaluronan in macrophage accumulation and collagen deposition after bleomycin-induced lung injury.

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.

Bleomycin-induced pulmonary injury in mice deficient in SPARC.

SPARC (secreted protein, acidic and rich in cysteine) is a component of the matrix that appears to regulate tissue remodeling. There is evidence that it accumulates in the lung in the setting of pulmonary injury and fibrosis, but direct evidence of its involvement is only now emerging. We therefore investigated the development of pulmonary fibrosis induced by bleomycin administered either intratracheally or intraperitoneally in mice deficient in SPARC. Bleomycin (0.15 U/mouse) given intratracheally induced significantly more pulmonary fibrosis in mice deficient in SPARC compared with that in wild-type control mice, with the mutant mice demonstrating greater neutrophil accumulation in the lung. However, in wild-type and SPARC-deficient mice given intraperitoneal bleomycin (0.8 U/injection x 5 injections over 14 days), the pattern and severity of pulmonary fibrosis, as well as the levels of leukocyte recruitment, were similar in both strains of mice. These findings suggest that the involvement of SPARC in pulmonary injury is likely to be complex, dependent on several factors including the type, duration, and intensity of the insult. Furthermore, increased neutrophil accumulation in the peritoneal cavity was also observed in SPARC-null mice after acute chemical peritonitis. Together, these data suggest a possible role for SPARC in the recruitment of neutrophils to sites of acute inflammation.

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment.

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.

Loss of endothelial surface expression of E-selectin in a patient with recurrent infections.

Neutrophil accumulation at sites of inflammation is mediated by specific groups of cell adhesion molecules including the beta2 (CD18) integrins on leukocytes and the selectins (P- and E-selectin on the endothelium and L-selectin on the leukocyte). This is supported by studies of patients with leukocyte adhesion deficiency syndromes whose leukocytes are genetically deficient in the expression of beta2 integrins or selectin carbohydrate ligands (eg, sialyl-Lewis(x)). However, inherited deficiency or dysfunction of endothelial cell adhesion molecules involved in leukocyte recruitment has not been previously described. In this report we describe a child with recurrent infections and clinical evidence of impaired pus formation reminiscent of a leukocyte adhesion deficiency syndrome, but whose neutrophils were functionally normal and expressed normal levels of CD18, L-selectin, and sialyl-Lewis(x). In contrast, immunohistochemical staining of inflamed tissue from the patient showed the absence of E-selectin from the endothelium, although E-selectin mRNA was present. However, E-selectin protein was expressed as significantly elevated levels of circulating soluble E-selectin were detected, the molecular size of which was consistent with a proteolytically cleaved form of E-selectin. Gene sequencing failed to show evidence of a secreted mutant variant. These data represent, to our knowledge, the first description of a potentially inherited dysfunction of an endothelial cell adhesion molecule involved in leukocyte recruitment and provide additional human evidence of the importance of endothelial selectins in the inflammatory response.

Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice.

Platelet endothelial cell adhesion molecule (PECAM-1/CD31), a member of the immunoglobulin superfamily expressed at high levels on endothelial cells, has been recently implicated in angiogenesis. Although antagonism of PECAM-1 inhibited neovascularization in two different animal models of growth factor/chemokine-induced angiogenesis, its participation in tumor angiogenesis has not been established. We therefore investigated its involvement in models of tumor angiogenesis in mice. An antibody against murine PECAM-1 that was shown to block in vitro murine endothelial tube formation inhibited the subcutaneous growth and tumor vascularity of three tumors in mice: A549 human non-small cell lung cancer in SCID mice, B16 murine melanoma in C57BL/6 mice and AB12 murine mesothelioma in Balb/c mice. These studies suggest a possible role for PECAM-1 in the complex process of tumor angiogenesis and provide additional evidence of the importance of endothelial cell adhesion molecules to the formation of new vessels.

Expression and function of endothelial cell alpha v integrin receptors in wound-induced human angiogenesis in human skin/SCID mice chimeras.

Accumulating evidence indicates that endothelial cell integrins that bind to the matrix proteins associated with inflammation and wound healing are involved in the process of angiogenesis. The integrins containing the alpha v subunit appear to be particularly important. To study the involvement of these receptors in human angiogenesis, a model of wound-associated human angiogenesis was established in human skin transplanted onto severe combined immunodeficient (SCID) mice. Using this model, we studied the expression of several alpha v integrins and tested the hypothesis that blockage of the alpha v beta 3 integrin would inhibit human angiogenesis during human wound healing. These studies revealed that the alpha v beta 3, alpha v beta 5, and alpha v beta 6 integrins are up-regulated briefly during wound angiogenesis with different patterns of expression and that inhibition of the alpha v beta 3 integrin blocked new vessel formation during human wound healing.

Tyrosine residue in exon 14 of the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates ligand binding specificity.

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19- amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell-cell aggregation while those variants missing exon 14 mediate homophilic cell-cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5-amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.

Involvement of endothelial PECAM-1/CD31 in angiogenesis.

The adhesive interactions of endothelial cells with each other and the adhesion receptors that mediate these interactions are probably of fundamental importance to the process of angiogenesis. We therefore studied the effect of inhibiting the function of the endothelial cell-cell adhesion molecule, PECAM-1/ CD31, in rat and murine models of angiogenesis. A polyclonal antibody to human PECAM-1, which cross-reacts with rat PECAM-1, was found to block in vitro tube formation by rat capillary endothelial cells and cytokine-induced rat corneal neovascularization. In mice, two monoclonal antibodies against murine PECAM-1 prevented vessel growth into subcutaneously implanted gels supplemented with basic fibroblast growth factor (bFGF). Taken together these findings provide evidence that PECAM-1 is involved in angiogenesis and suggest that the interactions of endothelial cell-cell adhesion molecules are important in the formation of new vessels.

Microvascular endothelium of human tumor xenografts expresses mouse (= host) CD31.

BACKGROUND: Human malignant tumors grown as xenografts in immunocompromised animals have been used extensively to study tumor growth and tumor response to therapy. The endothelium functions as an effective barrier between the intravascular space and the tumor cells. In a previous study we used species-specific monoclonal antibodies against endothelial cell adhesion molecules to demonstrate the host origin of the endothelium in xenotransplanted pancreatic islet grafts [Am J Pathol 1995;146:1397-1405]. We now investigated in this study whether the vascular endothelium of different xenografted human malignant tumors expresses mouse (= host)- or human (= graft)-specific CD31 (platelet endothelial cell adhesion molecule, PECAM-1) adhesion molecules. METHODS AND RESULTS: Cultured human prostate, kidney, and colon cancer cells (passages 15-17) were transplanted subcutaneously into 8-week-old athymic nude mice and removed after another 8 weeks. The avidin biotin peroxidase method was utilized on frozen sections to demonstrate that the endothelium of the vasculature of all three human xenografts expressed mouse (= host)-specific CD31, but not human (= graft)-specific CD31. CONCLUSION: The presence between the intravascular space and the human tumor cells of a mouse-derived endothelium, expressing mouse-specific antigens, needs to be taken into careful consideration when evaluating results of antitumor therapies in these animal models. This caveat pertains particularly to the study of novel cell- or tissue-specific treatment modalities, such as antibody-targeted drugs, toxins or radionuclides, 'immuno'-liposomes, or tumor vaccines.

Neutrophil platelet endothelial cell adhesion molecule-1 participates in neutrophil recruitment at inflammatory sites and is down-regulated after leukocyte extravasation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils, and has been shown to be involved in the migration of leukocytes across the endothelium. Although studies have supported a role for endothelial PECAM-1 in this process, the participation of neutrophil PECAM-1 in vivo has not been unambiguously demonstrated. Therefore, to examine the involvement of neutrophil PECAM-1 in leukocyte recruitment, we studied the effect of a blocking Ab against murine PECAM-1 on neutrophil recruitment in an established model of murine peritonitis and in a murine model for studying leukocyte-endothelial interactions involving the human vasculature. These studies not only confirmed that neutrophil PECAM-1 is important in the accumulation of neutrophils at inflammatory sites, but that extravasated neutrophils displayed decreased surface expression of PECAM-1. In vitro, the surface expression of murine neutrophil PECAM-1 was not decreased significantly by inflammatory mediators, but was reduced after transendothelial migration. These studies, consistent with previous in vitro observations, confirm that neutrophil PECAM-1, as well as endothelial PECAM-1, is involved in the recruitment of neutrophils into inflammatory sites in vivo, and suggest that the expression of neutrophil PECAM-1 is down-regulated after extravasation into inflamed tissues possibly as a result of engagement of its ligand.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1/CD31): A Multifunctional Vascular Cell Adhesion Molecule.

PECAM-1/CD31 is a member of the immunoglobulin gene superfamily found on platelets, leukocytes, and endothelial cells, where it concentrates at cell-cell borders. It has been shown to both mediate cell-cell adhesion through homophilic and heterophilic interactions and to transduce intracellular signals that upregulate the function of integrins on leukocytes. Its cellular distribution and ability to mediate adhesive and signaling phenomena suggested that PECAM-1 was a multifunctional vascular cell adhesion molecule involved in leukocyte-endothelial and endothelial-endothelial interactions. These initial suggestions have been largely confirmed as recent studies have implicated PECAM-1 in the inflammatory process and in the formation of blood vessels. As our understanding of the molecular and functional properties of PECAM-1 grows, new insights will be gained that may have therapeutic implications for cardiovascular development and disease. (Trends Cardiovasc Med 1997;7:203-210). © 1997, Elsevier Science Inc.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression.

PECAM-1/CD31 is vascular cell adhesion and signaling molecule of the Ig superfamily that plays a role in neutrophil recruitment at inflammatory sites and may be involved the release of leukocytes from the bone marrow and in cardiovascular development. The interactions of PECAM-1 with its ligands are complex in that it is able to bind both with itself (homophilic adhesion) or with non-PECAM-1 ligands (heterophilic adhesion). Although the factors that regulate ligand binding are not fully understood, these interactions are regulated in part by its large cytoplasmic domain, a region of 118 amino acids encoded by 8 exons of its gene (exons 9-16). The purpose of this work was to better define the mechanisms of PECAM-1-dependent homophilic adhesion by analyzing the binding interactions of L-cells expressing full-length and selectively mutated forms of human, murine, and human/murine chimeric PECAM-1 molecules in an established aggregation assay. These studies demonstrate that 1) the minimal length of the cytoplasmic domain required for cellular aggregation is represented within the sequences encoded by exons 9 and 10, 2) removal or addition of the sequences encoded by exon 14 from the cytoplasmic domain can determine whether the mechanism of aggregation is a heterophilic calcium-dependent process or a homophilic calcium-independent process, 3) high levels of surface expression of PECAM-1 on the cell surface change the mechanism of aggregation from heterophilic to homophilic, and 4) PECAM-1-dependent homophilic binding appears to involve the direct interaction of only the first two extracellular Ig-like domains. These data suggest that PECAM-1-ligand interactions can be regulated through multiple pathways including alterations of the cytoplasmic domain and the level of surface expression.

Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity.

PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown the PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane-proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells.

The role of selectins and CD18 in leukotriene B4-mediated white blood cell emigration in human skin grafts transplanted on SCID mice.

The purpose of this study was to examine the role of selectins and CD18 cell adhesion molecules (CAMs) in inflammation induced by injection of leukotriene B4 (LTB4) into human skin. To accomplish this, the expression of CAMs and the ability of specific antibodies against CAMs to block white blood cell (WBC) transmigration were studied in an in vivo model consisting of human skin transplanted onto mice with the severe combined immune deficiency (SCID) mutation. The results indicate that LTB4-induced WBC transmigration in the human/SCID model is rapid and pronounced; however, it is not accompanied by a significant upregulation of the baseline expression of endothelial P-selectin, E-selectin, ICAM-1 or VCAM-1. An anti-murine CD18 mAb markedly inhibited white cell infiltration (89% inhibition) confirming the importance of beta 2 integrins in the process. The role of selectins was also examined. MEL-14, a bioactive antibody against murine L-selectin inhibited transmigration by 66%. A significant, but smaller, effect (39% inhibition) was observed by blocking E-selectin function. These results indicate that LTB4-induced inflammation does not require upregulation of endothelial CAM expression and, in contrast to TNF alpha-induced transmigration, is only partially blocked by anti-E-selectin antibodies.