Vascular development and maintenance of proper vascular function through various regulatory mechanisms are critical to our wellbeing. Delineation of the regulatory processes involved in development of the vascular system and its function is one of the most important topics in human physiology and pathophysiology. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a cell adhesion molecule with proangiogenic and proinflammatory activity, has been the subject of numerous studies. In the present review, we look at the important roles that PECAM-1 and its isoforms play during angiogenesis, and its molecular mechanisms of action in the endothelium. In the endothelium, PECAM-1 not only plays a role as an adhesion molecule but also participates in intracellular signalling pathways which have an impact on various cell adhesive mechanisms and endothelial nitric oxide synthase (eNOS) expression and activity. In addition, recent studies from our laboratory have revealed an important relationship between PECAM-1 and endoglin expression. Endoglin is an essential molecule during angiogenesis, vascular development and integrity, and its expression and activity are compromised in the absence of PECAM-1. In the present review we discuss the roles that PECAM-1 isoforms may play in modulation of endothelial cell adhesive mechanisms, eNOS and endoglin expression and activity, and angiogenesis.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a 130-kDa transmembrane glycoprotein, is a member of the Ig gene superfamily. In the 1980s, PECAM-1 was characterized by a number of groups independently as a 130-kDa cell surface protein in the vascular compartment, which also includes endothelial cells (ECs), platelets and leukocytes, and identified as an EC junction molecule [1–5]. PECAM-1 is highly expressed on the surface of ECs and at lower levels in haematopoietic and immune cells, including macrophages, monocytes, neutrophils, natural killer cells, naive T-cells and B-cells . In cultured ECs, a large amount of PECAM-1 is expressed on the cell surface (∼106 molecules per cell ) and mainly localizes to sites of cell–cell contact . However, the exact role of PECAM-1, and more specifically its isoforms, in these activities is not fully understood.
The PECAM-1 gene, 65 kbp in length, is localized to the long arm of human chromosome 17 . Cloning of the human PECAM-1 gene demonstrated that the full-length human cDNA encodes an open reading frame of 738 amino acids consisting of a signal peptide (27 amino acids), an Ig-like extracellular domain (574 amino acids), a hydrophobic transmembrane domain (19 amino acids) and a relatively long cytoplasmic domain (118 amino acids), bearing multiple potential sites for phosphorylation and post-translational modifications such as carbohydrate and lipid modifications . The murine PECAM-1 cDNA was also cloned and it revealed 70–80% sequence homology with human PECAM-1 . Although the cytoplasmic domain of other cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), is encoded by a single exon, the PECAM-1 cytoplasmic domain is encoded by multiple exons. The PECAM-1 gene contains 16 exons with introns ranging in size from 86 bp to 12 000 bp; exons 1–2 encode 5′-UTRs and the signal peptide, exons 3–8 encode the six Ig-like extracellular domains, exon 9 encodes the hydrophobic transmembrane domain and exons 10–16 encode the long cytoplasmic domain .
The PECAM-1 gene produces a number of isoforms that differ only in the length of their cytoplasmic domain. These are generated by alternative splicing of exons 10–16. Although the PECAM-1 cytoplasmic domain lacks kinase activity, it participates in signalling cascades via regulated phosphorylation of specific tyrosine residues in exons 13 and 14. The expression of PECAM-1 isoforms in the vascular bed of various tissues, including the brain, heart, kidney, liver and lung from humans and mice, revealed that the alternative splicing of the human PECAM-1 gene is less frequent than that of the mouse one [11,12]. The alternative splicing of human PECAM-1 generates six isoforms including full-length, and those lacking exon 12, delta 13, delta 14, delta 15 and delta 14&15, and the full-length isoform is the major form of PECAM-1 in human tissues and ECs [12,13]. However, eight different PECAM-1 isoforms, including full-length, delta 12, delta 14, delta 15, delta 12&14, delta 12&15, delta 14&15, and delta 12, 14&15, are detected in most mouse tissues. In contrast to human tissues, delta 14&15 is the predominant isoform detected in various mouse tissues and ECs [11,13,14]. In addition, alternative splicing of PECAM-1 generates a number of these isoforms during differentiation and/or activation of haematopoietic cells and platelets [15,16].
Investigation of PECAM-1 isoform expression in mice demonstrated a regulated expression pattern during development [13,14,17], e.g. in the kidney, exon 14 expression was detected early in development, but not in the later stages . As each isoform has distinct cytoplasmic domains bearing different signalling potentials, PECAM-1 appears to have an impact on angiogenesis and/or vasculogenesis processes through alternative splicing of its cytoplasmic domain. However, elucidation of the physiological significance of these isoforms in the regulation of EC function remains very challenging and unexplored.
ECs line the inside of blood vessels and play critical roles in vascular function. EC precursors that arise from haematopoietic progenitors differentiate to ECs during vasculogenesis . Although ECs are sufficient for the formation and function of small blood vessels, larger blood vessels require EC interactions with pericytes or smooth muscle cells (SMCs) for vessel stabilization and integrity. In blood vessels, ECs exert their function through direct interactions with the bloodstream and various sensing stimuli such as hypoxia, growth factors, cytokines and shear stress. These activities are mediated through the production of a variety of mediators and modulation of intracellular signalling pathways, including those produced by PECAM-1 (Figure 1).
PECAM-1-mediated signal transduction
PECAM-1 SIGNAL TRANSDUSCTION
The human PECAM-1 cytoplasmic domain contains 12 serine, 5 threonine and 5 tyrosine residues, and serine/threonine residues in the cytoplasmic domain of PECAM-1 are constitutively phosphorylated [19,20]. However, the phosphorylation of tyrosine residues in exon 13 (Tyr663) and exon 14 (Tyr686) are actively regulated by protein tyrosine kinases (PTKs, e.g. the Src and Csk family of kinases) and SHP-2 phosphatase [21,22]. These tyrosine phosphorylations are modulated in response to physiological stimuli, including platelet aggregation , engagement of the high-affinity IgE receptor on basophils and mast cells , triggering of the antigen receptor on T-cells  and mechanical stimulation of ECs [26,27]. The distinct cellular status of ECs exhibits different levels of PECAM-1 phosphorylation, decreased phosphorylation in migrating cells and maximum phosphorylation in attached confluent ECs . Examination of the regulation of the PECAM-1 cytoplasmic domain in its junctional localization has revealed that deletion of this domain results in a lack of cell–cell junctional localization in mice fibroblast L-cells [29,30]. Thus, it is now becoming apparent that intracellular signalling for cell migration and formation of cell–cell junctions is regulated by phosphorylation of the PECAM-1 cytoplasmic domain [22,31].
The PECAM-1 cytoplasmic sequence expansion of Tyr663 and Tyr686 shares common features of immunoreceptor tyrosine-based inhibitory motifs [32–34], which on phosphorylation exert an inhibitory function by recruiting and activating protein tyrosine phosphatases, including SHP-1 and SHP-2 [35–38]. Transient over-expression of mouse PECAM-1 in COS cells demonstrated that phosphorylation of PECAM-1 by Src family kinases was sufficient to promote binding of SHP-1 and SHP-2 . Thus, phosphorylated tyrosine residues in the PECAM-1 cytoplasmic domain provide docking sites for SH2 domain-containing signalling molecules, predominantly SHP-2, and bound SHP-2 phosphatase inhibits the PECAM-1-mediated signalling cascade . Lack of phosphorylation of PECAM-1 results in failure to recruit SHP-2 phosphatase and enhanced mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling in ECs, which results in enhanced EC migration and abrogated formation of cell–cell adherens junctions  (Figure 2). Thus, the regulation of SHP-2 phosphatase activity is essential in PECAM-1-mediated, cellular signalling cascades, especially MAPK/ERK, which have an impact on EC proliferation, survival, differentiation and migration (see Figure 1) [32,40–43].
PECAM-1-mediated signalling pathways through interactions between phosphorylated tyrosine residues in the cytoplasmic domain and SHP-2
The PECAM-1 cytoplasmic domain contains tyrosine residues in exon 13 (Tyr663) and exon 14 (Tyr686) and alternative splicing of this domain could produce isoforms with or without exon 14 (Tyr686) [11–14]. As a result, each isoform has a different binding capacity for SH2 domain-containing signalling molecules. PECAM-1 isoform-expressing Madin–Darby canine kidney (MDCK) cells revealed that interaction between PECAM-1 and Src kinase is exon 14 independent, whereas SHP-2 association is exon 14 dependent . Investigation of PECAM-1 isoform-specific functions in signalling pathways was conducted by re-expressing the delta 15 or delta 14&15 PECAM-1 isoform in MDCK epithelial cells lacking PECAM-1 expression or PECAM-1-deficient (PECAM-1−/−) brain endothelial (bEND) cells–a polyoma middle T-transformed line of brain ECs prepared from PECAM-1−/− mice [39,44,45]. In PECAM-1−/− bEND cells, the phosphorylation level of the delta 15 PECAM-1 isoform was higher than that of the delta 14&15 isoform, and consequently the activation level of MAPK/ERK in delta 15 isoform-expressing cells was comparable with parental PECAM-1−/− cells . In contrast, delta 14&15 PECAM-1 isoform expression enhanced MAPK/ERK activation and resulted in abrogated formation of cell–cell adherens junctions and enhanced migration of PECAM-1−/− bEND cells [39,44,45]. Furthermore, re-expression of isoforms lacking exon 14 (delta 14 or delta 14&15) in PECAM-1−/− kidney and retinal ECs restored the proangiogenic properties, including migration, capillary morphogenesis and cell adhesion to extracellular matrix (ECM) proteins [45,46]. Taken together, exon 14-dependent SHP-2 association with the PECAM-1 cytoplasmic domain makes a significant contribution to signalling pathways having an impact on angiogenesis. This may occur through competition between SHP-2 and Src for Tyr663 (exon 13).
VASCULAR FUNCTION OF PECAM-1
Nutrients and oxygen are provided to the organs by blood circulation. Accordingly the development of the vascular network is one of the most critical events in embryogenesis. The development of vasculature fundamentally occurs via three processes: vasculogenesis, angiogenesis and vascular remodelling. Vasculogenesis refers to the new vascular network formed by differentiated ECs from vascular endothelial precursor cells termed ‘angioblasts’ . During early embryogenesis, the primary vasculature is formed by vasculogenesis. Angiogenesis is the process by which blood vessels are formed from pre-existing capillaries, and mediates vascular development until the vasculature has been formed . After completion of development, angiogenesis is restricted to just the ovarian cycle and the placenta during pregnancy . However, some physiological stimuli reactivate angiogenesis in adulthood such as wound healing and hypoxia .
Angiogenesis is tightly regulated by a balanced production of inhibitory [e.g. pigment epithelium-derived factor (PEDF), thrombospondin-1 (TSP1), TSP2, angiostatin, endostatin] and stimulatory [e.g. vascular endothelial growth factor (VEGF) family, fibroblast growth factor (FGF) family, epidermal growth factor (EGF), PECAM-1] factors . Unregulated angiogenesis is involved in more than 70 disorders including cancer, inflammatory disorders, obesity, asthma, diabetes, autoimmune diseases and various eye diseases [18,50]. These are generally associated with increased production of proangiogenic factors and decreased production of anti-angiogenic factors, which tip the angiogenic balance towards angiogenesis.
Vascular remodelling is an adaptive structural alteration occurring in response to long-term changes in haemodynamic conditions. The process is modulated by locally generated growth factors, vasoactive substances and haemodynamic stimuli, and is accomplished by changes in cellular processes including cell growth, cell death, cell migration, and production or degradation of the ECM . Our studies of retinal postnatal vascular development have demonstrated an important role for TSP1, a matricellular protein with anti-angiogenic activity, in retinal vascular maturation .
Endothelial cells and angiogenesis
Vessel formation is initiated by the production of angiogenic growth factors, including VEGF, placental growth factor, angiopoietin-1, inhibitors of differentiation proteins and cytokines [54–57]. After binding to their respective receptors on ECs, these factors promote EC proliferation, migration and capillary morphogenesis, which are stabilized by recruitment and interaction with pericytes or SMCs. In the process of vessel formation, ECs' distinctive features including cell migration and capillary morphogenesis have an essential role to play. Capillary morphogenesis refers to the process of forming tube-like networks between ECs, which is a unique and pivotal feature of these cells. Migration is regulated by the interaction between integrins on the EC surface and the ECM proteins that are produced by ECs, pericytes and SMCs, filling the extracellular space. Integrins, receptors for ECM proteins, and the Ig superfamily of cell adhesion molecules mediate cell migration through activation of intracellular signalling pathways including focal adhesion kinase (FAK), Src and many other kinases . Through the formation of functional actin filament and focal adhesions, ECs migrate with directivity towards the source of promigratory signals. The role of PECAM-1 in these activities and how these activities are impacted by various isoforms of PECAM-1 need further investigation.
PECAM-1 in vascular development and remodelling
To investigate the role of PECAM-1 in vasculogenesis and angiogenesis further, Duncan et al.  disrupted the PECAM-1 gene in embryonic stem cells to generate PECAM-1−/− mice, which have been reported to be viable and born without critical vascular defects . However, later they were shown to exhibit attenuated alveolarization, an angiogenesis-dependent process during lung development , postnatal retinal vascular development and brain angiogenesis abnormalities [61–63]. In addition, PECAM-1−/− mice showed decreased retinal vascular density, abnormal secondary branch formations and increased vessel diameter, and also failed to undergo neovascularization during oxygen-induced ischaemic retinopathy . These observations suggest an important role for PECAM-1 in normal vascular development and angiogenesis, and potential compensatory mechanisms that may minimize embryonic defects in vivo.
The formation of a functional vessel requires proper remodelling in response to a chronic stimulus on vessel walls. During vascular remodelling, ECs sense the stimulus on vessel walls and, accordingly, vessels enlarge or reduce the lumen size. The blood flow is one of the important stimuli for vascular remodelling . An important role of PECAM-1 in sensing fluid shear stress (FSS) has been addressed by defining PECAM-1 as a major component of the mechanosensory complex sensing shear stress [27,64] through changes in its phosphorylation [26,65,66]. Actually previous studies demonstrated that PECAM-1−/− mice are defective in responding to shear stress-mediated vascular remodelling , and collateral vessel formation, the latter being more dilated and shorter than those in wild-type mice .
PECAM-1 and inflammation
The vascular endothelium is important not only in angiogenesis but also in immune responses. During immunosurveillance and inflammation, leukocytes migrate from the bloodstream to targeted tissues. Transmigration of leukocytes through the endothelium is a critical step accomplished via the interaction of cell adhesion molecules between leukocytes and ECs and/or between ECs. The multistep leukocyte trafficking process, named transendothelial migration (TEM), is conducted through well-established sequential steps of leukocyte rolling, activation, adhesion and migration through the endothelium. As leukocyte migration is mediated by the interaction between the active receptor on the leukocytes and ligands on the ECs, the expression and activation of adhesion molecules are critical determinants of this process . During the leukocyte rolling step, interactions with selectins and glycosylated ligands slow their migration . Activated leukocytes exposed to chemokines then enhance integrin activity and increase their adhesion to cell adhesion molecules, members of the Ig superfamily including PECAM-1, ICAM-1, ICAM-2 and VCAM-1 . Loosening of cell–cell adhesions between ECs by the disruption of the homophilic interactions of adhesion molecules [e.g. junctional adhesion molecule (JAM)/JAM and PECAM-1/PECAM-1), results in diapedesis of leukocytes and completion of TEM. Recent studies also demonstrate an active role for perivascular supporting cells in these activities . However, the contribution of PECAM-1 to prevascular supporting cells' activities during inflammation remains unknown.
Muller et al.  suggested a role for PECAM-1 in leukocyte transmigration through the observation that PECAM-1-specific antibodies inhibited leukocyte transmigration through the endothelium. Additional studies have now demonstrated a critical role for PECAM-1 in leukocyte transmigration in vivo [74–76] and in vitro [75,77,78]. In PECAM-1−/− mice, the absence of PECAM-1 resulted in attenuation of leukocyte TEM, neutrophil recruitment and inflammatory responses [59,79]. However, there are contrasting studies indicating minimal involvement of PECAM-1 in the transmigration of leukocytes , and mice strain-specific defects with PECAM-1 deficiency may be a contributory factor . Furthermore, transmigration of leukocytes is not necessarily undesirable, e.g. Perkowsky et al.  showed that inhibition of this reaction by blocking PECAM-1 function does not alleviate pulmonary oedema.
PECAM-1 expression on ECs and leukocytes mediates the homophilic interactions between ECs and leukocytes and between ECs themselves. During transmigration, PECAM-1 expressed on leukocytes contributes to chemokine-mediated directional migration of leukocytes to the sites of inflammation . After leukocyte adhesion to the endothelium, PECAM-1 homophilic interactions between leukocytes and ECs promote leukocyte migration through cell–cell junctions [83,84] and enhance the activation of integrins on leukocytes, which promotes migration across the perivascular basement membrane [6,59,74,84]. A number of studies have suggested that the function of PECAM-1 in this process is independent of its SHP-2 recruitment [85,86]. However, the detailed signalling pathways involved and the potential contribution of perivascular supporting cells to these activities require further delineation.
HOMOPHILIC AND HETEROPHILIC INTERACTIONS OF PECAM-1
The PECAM-1 Ig-like homology domain of the extracellular region exhibits similar characteristics to other Ig superfamily members, and plays an important role in cell adhesion through homophilic binding of PECAM-1 [3,87,88] and heterophilic binding [89,90] of PECAM-1 and other cell surface molecules, including αvβ3 [91–93], CD38 , the 120-kDa ligand on T-cells  and heparin-dependent proteoglycans . Previous studies have demonstrated that the PECAM-1 cytoplasmic domain modulates its heterophilic and homophilic binding characteristics [96,97]. Deletion of the entire PECAM-1 cytoplasmic domain impairs PECAM-1-mediated cell–cell interactions, whereas its partial deletion has a differential impact on these activities . Furthermore, PECAM-1 isoforms showed different cell aggregation capacity depending on the presence or absence of exon 14 in their cytoplasmic domain. Although isoforms containing exon 14 showed heterophilic, calcium-dependent and heparin-sensitive aggregation, isoforms lacking exon 14 exhibited homophilic, calcium-independent and heparin-insensitive aggregation [29,98]. Thus, PECAM-1-mediated cell–cell interactions through heterophilic and homophilic interactions are regulated by the presence or absence of exon 14. However, most of these studies were conducted in non-ECs, which lacked PECAM-1 expression, and perhaps other EC-specific molecules that may have an impact on PECAM-1 function. Therefore, the adhesion properties of these isoforms in ECs lacking PECAM-1 are of great interest and should provide important insight into their activities.
PROANGIOGENIC ROLES OF PECAM-1
Cell–cell junction formation
In humans a number of pathological conditions, including ischaemic stroke and inflammation, are due to defects in endothelial permeability. Endothelial monolayers form cell–cell junctions through the interactions of adhesion molecules on ECs, and disruption of the cell–cell junction causes altered vascular permeability and fragility. Numerous genes involved in vascular remodelling and vessel integrity have been identified, and their deletion or down-regulation results in embryonic lethality caused by defective vascular development .
In the endothelium, adhesion and communication between ECs are mediated by intercellular junctions including adherens junctions (AJs), tight junctions (TJs) and gap junctions (GJs). As a communication structure, GJs provide the passage for low-molecular-mass solutes between neighbouring cells. In ECs, AJs and TJs are major types of junctions [99–101] and the functions of AJs and TJs are distinctive. AJs are involved in the initiation of cell–cell contacts and their maturation, and TJs participate in the passage of ions and solutes. Through the formation of intercellular junctions, ECs sense their position, regulate cell growth and apoptosis, and develop tubular structures [102–104]. Junction formation in ECs is mediated by the homophilic bindings of transmembrane proteins, including VE-cadherin, catenin, ZO-1 and PECAM-1 for AJs, and occludins, claudins and ZO-1 for TJs .
The observation of increased vascular permeability in PECAM-1−/− mice supports a role for PECAM-1 in AJ formation and function . In cultured ECs, PECAM-1 localizes at the edge of cells together with other proteins, and cell confluency enhances its junctional localization [1,46,105]. Incubation of bovine ECs with anti-PECAM-1 antibody revealed that PECAM-1 functions to establish and maintain cell–cell junctions. In this study, junction formation was affected when ECs were incubated with PECAM-1 antibody before forming a confluent EC monolayer; however, after complete junction formation, the PECAM-1 antibody did not influence cell–cell junctions . Additional insight into the role of PECAM-1 isoforms in cell–cell junction formation was investigated by expressing a specific PECAM-1 isoform in MDCK cells, which similar to the ECs form various types of junctions but lack PECAM-1 expression, and PECAM-1−/− bEND cells prepared from PECAM-1−/− mice [39,40]. These studies indicated, for the first time, that junction formation is actively modulated by PECAM-1 in an isoform-specific manner through modulation of intracellular signalling pathways, and junctional localization of PECAM-1 is dependent on formation of AJs as discussed above.
EC migration and adhesion
EC migration during angiogenesis is conducted by three major mechanisms: (i) chemotaxis, migration towards a gradient of chemoattractants; (ii) haptotaxis, migration towards a gradient of immobilized ligands; and (iii) mechanotaxis, migration mediated by mechanical forces . Although growth factors such as VEGF and basic fibroblast growth factor (bFGF) induce chemotactic migration of ECs, binding of integrins to the ECM is involved in haptotaxis of ECs [41,107]. To migrate, the cytoskeleton needs to undergo constant remodelling into filopodia, lamellipodia and formation of actin stress fibres.
The first indication that PECAM-1 regulates cell migration was demonstrated with the expression of full-length PECAM-1 in NIH3T3 cells, a line of mouse fibroblasts that normally lack PECAM-1 expression . NIH3T3 cells expressing full-length PECAM-1 showed enhanced cell adhesion and reduced cell migration. Since then numerous studies have used PECAM-1 antibodies to demonstrate the role of PECAM-1 in EC migration, and incubation with PECAM-1 antibodies inhibited EC migration [60,63,109] and capillary morphogenesis [109–114]. More specific understanding of the role of PECAM-1 in EC migration was provided by studying isolated ECs from PECAM-1−/− mice. Lack of PECAM-1 in ECs resulted in abrogated cell migration [46,63,115] and capillary morphogenesis [46,116]. In contrast, enhanced PECAM-1 expression increased EC motility [46,109,111]. Reduced PECAM-1−/− EC migration was characterized by defective remodelling of the cytoskeleton into filopodia and lamellipodia, and enhanced adhesion to ECM proteins [46,63].
PECAM-1 modulates EC migration by its impact on intracellular signalling cascades through phosphorylation of its cytoplasmic tyrosine residues. The expression of tyrosine residue-mutated or cytoplasmic domain-truncated PECAM-1 in ECs increased the migration and phosphorylation of other migration regulators, including β-catenin and FAK [97,105,117]. Recent studies demonstrated that loss of exon 14 enhances migration by activation of MAPK/ERK signalling and decreasing cell adhesion to ECM protein [41,44,45,117]. This isoform-specific effect on cell migration provides meaningful insight into understanding of the differential role that PECAM-1 isoforms may play during tissue development [13,14,17].
In the endothelium, the confluent monolayer of ECs remains in a contact-inhibition state, which limits their proliferation and migration, and PECAM-1 may exert a regulatory function in this process . Proliferation of ECs is a critical process during angiogenesis, which is promoted by proangiogenic growth factors including VEGF and bFGF. The role of PECAM-1 in angiogenesis has been approached by applying blocking antibodies against PECAM-1 in vivo, and the investigations revealed abrogated, cytokine-induced, rat corneal angiogenesis, bFGF-induced angiogenesis in Matrigel implant assays  and tumour angiogenesis [112,119]. In addition, cell proliferation in the retinal vasculature was enhanced in PECAM-1−/− mice . However, the effect of PECAM-1 on EC proliferation in vitro varies depending on the source of ECs, and the methods used to down-regulate PECAM-1 function or expression (e.g. PECAM-1-specific antibodies and siRNAs). PECAM-1+/+ and PECAM-1−/− lung ECs showed comparable levels of proliferation , whereas PECAM-1−/− bEND cells and retinal ECs (both from the central nervous system) were more proliferative compared with PECAM-1+/+ cells [46,115]. Thus, the role of PECAM-1 in EC proliferation is tissue specific and the PECAM-1 deficiency level effects on the proliferating process may be linked to expression and/or activity of CD44 through the Hippo signalling pathway [120,121].
PECAM-1 and endothelial targeting
The specific expression of high levels of PECAM-1 in the endothelium has been utilized as a potential target for specific delivery of various materials with therapeutic potential [122,123]. Muzykantov et al.  initially showed that clustering of biotinylated anti-PECAM-1 antibody with streptavidin significantly enhanced uptake and internalization of anti-PECAM-1 antibody by cells expressing PECMA-1. Furthermore, conjugation of catalase with biotinylated streptavidin meant that the anti-PECAM-1 antibody was taken up by ECs which protected them from hydrogen peroxide-mediated injury, both in culture and in perfused lungs . Using a similar strategy they later showed the utility of this immunotargeting in clinical lung transplantation and perhaps other disorders with an endothelial component [123–131]. These studies also led to identification of a novel endocytic pathway for anti-PECAM-1 antibody conjugates independent of clathrin-mediated or caveolar endocytosis . However, agents that inhibit macropinocytosis resulted in reduced internalization of clustered PECAM-1. Additional studies demonstrated an important role for the PECAM-1 cytoplasmic domain including Tyr686 for rho activation and actin polymerization, and endocytosis of anti-PECAM-1 antibodies . Thus, PECAM-1 signalling and interaction with the cytoskeleton provide a safe intra-endothelial drug delivery for anti-PECAM-1 carriers, and may be influenced by distinct, targeted, PECAM-1 epitopes .
PECAM-1, eNOS AND REGULATION OF NO PRODUCTION
Biology of eNOS
Nitric oxide (NO) is a short-lived signalling molecule participating in the regulation of vascular tone, vascular remodelling, endothelial permeability and angiogenesis [135–137]. Nitric oxide synthase (NOS) catalyses NO production from L-arginine. There are three main isoforms of NOS: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and eNOS (or NOS3). The eNOS/NOS3 is the predominant NOS isoform in ECs. During angiogenesis, NO plays a role through the regulation of physiological processes, including cell survival, proliferation and migration. The activity of eNOS is modulated by the phosphorylation of Ser1177 (activation) and Thr495 (inhibitory)  and dephosphorylation of Ser113, which is known to be involved in VEGF-mediated NO production [139,140]. Thus, post-translational modifications of specific serine and threonine residues have a high impact on eNOS activity.
ECs line the inside of blood vessels and are consequently exposed to the FSS of the bloodstream, which has an impact on EC morphology, gene expression and physiology . FSS is the most essential physiological stimulus for eNOS activation and NO production in the endothelium [141–143]. Although eNOS localizes to the perinuclear region, specifically the Golgi body , under subconfluent conditions the main localization of eNOS in confluent monolayers is at sites of cell–cell contact [135,145,146]. As NO functions to regulate the permeability of the endothelium by responding to stimuli, the presence of eNOS at cell–cell contact sites is critical for its activation .
Caveolae, plasma membrane structures shaped like a cavity, are a site enriched with various signalling molecules, ion channels, eNOS and protein kinases [143,147–149], and are a place where eNOS is activated after FSS . Noel et al.  demonstrated an important role for PECAM-1 and caveolae in enhanced angiogenic signalling in pulmonary endothelium after a stopped flow challenge. These studies demonstrated that PECAM-1 and caveolae are parts of the mechanosensing complex that modulates NOX2 activation and production of superoxides, which promote neutrophil influx and proangiogenic signalling with loss of shear. These activities were attenuated in the absence of PECAM-1, perhaps as a result of a failure to sense loss of shear and the need for PECAM-1 in neutrophil infiltration. However, the detailed mechanisms involved in PECAM-1 caveolar localization, and the potential role that PECAM-1 isoforms and eNOS interactions may have in the formation of the mechanosensing complex and these activities, remain to be determined.
As indicated above, antibody to PECAM-1 has provided a suitable carrier for delivery and uptake of various agents to the endothelium. The impact of shear stress on ECs may also have an impact on their interactions with and uptake of multivalent nanocarriers coated with PECAM-1 antibodies. These carriers are taken up via a non-canonical endocytic pathway that is related to macropinocytosis . Han et al.  showed that EC chronic adaptation to flow inhibited the endocytosis of nanocarriers coated with PECAM-1 antibody in arterial compared with capillary vessels. However, acute flows without stress fibre formation stimulated the endocytosis of nanocarriers incubated with PECAM-1. Furthermore, deletion of the PECAM-1 cytoplasmic domain and disruption of cholesterol-rich plasmalemma domains attenuated the stimulation of endocytosis of nanocarriers incubated with PECAM-1 by acute flow. Thus, the local microenvironment has a significant impact on the uptake of nanocarriers by the endothelium, and cell culture models of nanoparticle uptake should reflect the microenvironment and phenotype of target cells. In addition, co-administration of a paired monoclonal antibody directed to an adjacent, yet distinct, PECAM-1 epitope increased the binding of PECAM-1-directed antibodies with significant enhancement of functional activity of the nanocarriers .
PECAM-1 deficiency and eNOS
Bagi et al.  showed that PECAM-1−/− mice exhibit defective vasodilatory activities in their arteries, which suggested a critical role for PECAM-1 in the regulation of eNOS activity and NO production. Dilated retinal vessels in PECAM-1−/− mice provide further insight into the role of PECAM-1 in NO production . The retinas of PECAM-1−/− mice expressed lower levels of eNOS  and retinal ECs from PECAM-1−/− mice expressed a significantly reduced level of eNOS and its phosphorylated active form; as a result there is a dramatic decrease in NO production . However, the effect of PECAM-1 deficiency on eNOS expression is tissue dependent. Aortas from PECAM-1−/− mice exhibited comparable levels of eNOS to those in PECAM-1+/+ mice  and PECAM-1−/− bEND cells expressed higher levels of eNOS than PECAM-1+/+ bEND cells (Figure 3). Although the eNOS expression level was not down-regulated as a result of the lack of PECAM-1, PECAM-1−/− bEND cells were defective in NO production (Figure 3). Thus, PECAM-1 expression may contribute to appropriate activation of eNOS.
Activation of eNOS in PECAM-1+/+ and PECAM-1−/− bEND cells
As explained above, eNOS activity is regulated by phosphorylation and dephosphorylation of several sites on eNOS (e.g. Ser1177, Thr495 and Ser113), and PECAM-1 appears to be involved in the regulation of these phosphorylation and dephosphorylation processes. A lack of PECAM-1 did not affect the phosphorylation of Ser1177, the phosphorylation of which activates NO production. However, increased phosphorylation levels of Thr495 (inhibitory site) and Ser113, the dephosphorylation of which is critical in VEGF-mediated NO production [139,140], were observed in PECAM-1−/− bEND cells. This altered phosphorylation status seems to contribute to defective NO production in PECAM-1−/− bEND cells (Figure 3), and deserves further investigation.
PECAM-1−/− retinal ECs exhibited abrogated NO production as a result of the down-regulated eNOS expression level . It is interesting that decreased NO production was observed in PECAM-1−/− bEND cells, which expressed even higher level of eNOS and comparable levels of Ser1177 phosphorylation (Figure 3). These results indicated that PECAM-1 is involved in NO production of ECs by regulating not just eNOS expression but also phosphorylation for activation through undetermined processes.
PECAM-1-mediated regulation of eNOS activity
A considerable number of studies have demonstrated various FSS sensors, including ion channels, tyrosine kinase receptors, G-protein-coupled receptors, caveolae, adhesion molecules including integrins, PECAM-1, glycocalyx and primary cilia . In confluent ECs, the monolayer PECAM-1 localizes at sites of cell–cell contact, and application of FSS in these cells induces phosphorylation of tyrosine residues in the PECAM-1 cytoplasmic domain [26,66,153,154], which mediates ‘mechanosignal transduction’ through activation of MAPK/ERK and recruitment of SHP-2 [23,155].
Phosphorylation of tyrosine residues in PECAM-1 on FSS induces phosphorylation and activation of eNOS. The down-regulation of PECAM-1 expression in ECs induces defective phosphorylation of eNOS . Moreover, recent studies have demonstrated that PECAM-1 is important in the activation of eNOS through direct protein–protein interactions at cell–cell junctions [135,152,156,157] (Figure 4). These studies indicated that PECAM-1 may play a role in restricting eNOS localization to cell–cell junctions, and PECAM-1 association with eNOS results in its inactivation. After the phosphorylation of PECAM-1 on FSS, the association of PECAM-1 and eNOS is disrupted, and the dissociation enhances eNOS activity [156,158]. Thus, PECAM-1 is implicated in the mechanotransduction and regulation of eNOS activity through its direct association at cell–cell contacts. However, the detailed mechanisms involved, and whether these activities have an impact in a PECAM-1 isoform-specific manner, remain unclear and are an active area of investigation.
Proposed regulation of eNOS activity through interactions with PECAM-1
PECAM-1 isoforms and caveolar localization of eNOS
In cell membranes, a number of molecules including PECAM-1 and eNOS are enriched in caveolae for efficient interaction and signalling. It has been demonstrated that endothelial junctional localization of eNOS is modulated by its interaction with PECAM-1 [135,156,158]. However, our understanding of the impact of PECAM-1 isoforms on eNOS activity is quite limited. To determine the effect of PECAM-1 isoforms on caveolar localization of eNOS, each PECAM-1 isoform-expressing EC was analysed after caveolar fractionation on ultracentrifugation. As PECAM-1−/− retinal ECs express significantly decreased levels of eNOS compared with PECAM-1+/+ ECs , the former were transfected with a specific isoform of PECAM-1 and eNOS for these assays. After fractionation on ultracentrifugation, caveolar fractions were examined for caveolin-1, a protein component of the caveolar structure which was mainly present in fraction 5 (Figure 5A). In PECAM-1+/+ retinal ECs, both PECAM-1 and eNOS were predominantly localized in fraction 5 (Figure 5). Expression of PECAM-1 isoforms with and without exon 14 did not affect PECAM-1 and eNOS caveolae localization in ECs (Figures 5B and 5C). The eNOS, in PECAM-1+/+ ECs, was mainly localized to caveolae (fraction 5), but eNOS in PECAM-1−/− ECs expressing a specific isoform of PECAM-1 exhibited a more diffuse localization, such as the Golgi body (fractions 6–8), endoplasmic reticulum, microsomes (fractions 9–11) and nuclear pellet (fraction 12) (Figure 5). Analysis of PECAM-1 isoform-expressing PECAM-1−/− bEND cells, which do express significant amounts of eNOS, exhibited a similar result (not shown). Thus, caveolar localization of PECAM-1 appears to be isoform independent, and altered localization of eNOS in PECAM-1−/− ECs expressing a specific PECAM-1 isoform requires further investigation and may be influenced by FSS.
PECAM-1 is not sufficient for caveolar localization of eNOS
Isoform-specific interaction of PECAM-1 and eNOS
PECAM-1 is known to modulate eNOS activity through protein–protein interactions at sites of cell–cell contact [135,156,158]. However, the role of PECAM-1 isoforms in this process is still unclear. To enhance our understanding of this process, the PECAM-1 isoform-specific interaction with eNOS in PECAM-1−/− bEND cells expressing a specific isoform of PECAM-1 was analysed by immunoprecipitation (IP) assays. Among various isoforms, delta 15 and delta 14&15 isoforms showed intense interaction with eNOS (Figure 6A). However, the relative amount of PECAM-1 bound to eNOS was significantly lower than free eNOS (Figure 6A; ∼10%: IP vs lysate). Also, PECAM-1-isoform expression in PECAM-1−/− cells did not affect the abrogated NO production (Figure 6B) and phosphorylation of eNOS (not shown). Here the experiments were performed under normal culture conditions with ECs isolated from retinas and brain microvessels. These results suggested that interaction between PECAM-1 and eNOS in microvessels may not be the major step in control of NO production, and FSS might be the most potent stimulator of the PECAM-1 and eNOS interaction. Therefore, investigation of applied shear stress to PECAM-1−/− ECs expressing a specific isoform of PECAM-1 may provide a better understanding of the PECAM-1 isoform-specific interaction with eNOS and its implication in EC function.
PECAM-1 isoform-specific interaction with eNOS and the effect of PECAM-1 expression on NO production
PECAM-1 AND ENDOGLIN
Biology of endoglin
Endoglin (CD105) is a 180-kDa, homodimeric, transmembrane glycoprotein that functions as an auxiliary receptor in transforming growth factor β (TGF-β) signalling. After its initial identification in pre-B-lymphoblastic HOON cell lines , expression of endoglin on vascular ECs, acute lymphoblastic and myelocytic leukaemia cells, and bone marrow cells was reported [159–161]. Loss of endoglin is embryonically lethal due to defects in cardiovasculature development [162,163]. Heterozygous mutations in the external domain of endoglin results in haploinsufficiency, and is implicated in haemorrhagic telangiectasia type 1 (HHT1) and arteriovenous malformations in humans . In mice, endoglin haploinsufficiency also results in vascular defects similar to HHT1 .
Endoglin participates in angiogenesis through regulation of EC proliferation, migration and capillary morphogenesis [165,166]. Based on the observations of highly increased endoglin expression in active ECs on tumour angiogenesis [167,168], the development of cancer therapy targeting endoglin is under intense investigation . These activities of endoglin are mediated through both canonical and non-canonical TGF-β signalling pathways. However, the detailed implication of endoglin in these mechanisms still remains poorly understood.
Endoglin and TGF-β signalling pathways
TGF-β is a cytokine with an important role in EC proliferation, differentiation, migration and survival [170–172], and includes three isoforms: TGF-β1, TGF-β2 and TGF-β3. TGF-β is shown to both inhibit and stimulate angiogenesis depending on experimental conditions [173,174]. The main function of TGF-β is mediated through tyrosine/threonine kinase receptors on the cell surface including TGF-β type II receptor (TGF-β-RII), TGF-β type I receptor (TGF-β-RI) and endoglin . There are two distinct types of TGF-β-RI including activin receptor-like kinase 1 (ALK1), which is exclusively expressed on ECs, and ALK5 with a more universal expression. After TGF-β ligand binding to TGF-β-RII, TGF-β-RI is recruited and a heteromeric complex of two TGF-β-RII and two TGF-β-RI receptors is formed. TGF-β-RII then phosphorylates TGF-β-RI, which mediates the signalling cascade by phosphorylating the intracellular effector proteins, Smads [176,177]. TGF-β is known to participate in angiogenesis by stimulating or inhibiting the activation of ECs through a balance of ALK5 and ALK1 signalling. Although activated ALK5 phosphorylates Smad2/3 and induces quiescence of ECs, phosphorylation of Smad1/5/8 after activation of ALK1 activates ECs to migrate and proliferate (Figure 7) .
The proposed role of endoglin in TGF-β signalling pathways
Endoglin, as an auxiliary TGF-β-R, is a component of the TGF-β-R complex system and binds TGF-β1 and TGF-β3 [179,180]. Activated ALK5, ALK1 and TGF-β-RII interact with endoglin and phosphorylate serine and threonine residues in the endoglin cytoplasmic domain; the phosphorylation of endoglin by an endothelial-specific ALK1 regulates ALK1-dependent EC growth and adhesion [181,182]. TGF-β also regulates angiogenesis through non-Smad-dependent signalling pathways, including MAPK, rho GTPase and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathways [183,184]. How these signalling pathways contribute to the promotion and/or inhibition of angiogenesis needs further elucidation.
Endoglin and PECAM-1
Endoglin expression is highly up-regulated during angiogenesis. PECAM-1−/− mice exhibited defects in angiogenesis perhaps due to reduced endoglin expression and failure to up-regulate endoglin expression during acute neovascularization . There is very limited knowledge about the relationship between PECAM-1 and endoglin, and the effect of a PECAM-1 deficiency on endoglin expression and function. In one study altered angiogenic properties and the TGF-β signalling cascade of retinal ECs prepared from endoglin+/− mice were observed; this study was the first to delineate the cell's autonomous impact of endoglin haploinsufficiency on EC functions . It revealed an interesting correlation between PECAM-1 and endoglin expression which reduced endoglin expression, causing down-regulation of PECAM-1. Through undetermined mechanisms, PECAM-1 and endoglin influence each other's expression, and their altered expressions generate defective angiogenic properties of ECs through intracellular signalling pathways on which TGF-β has an impact. However, the detailed regulatory mechanisms engaged during angiogenesis, which result in proper expression of endoglin, need further elucidation.
CONCLUSIONS AND FUTURE DIRECTIONS
PECAM-1 not only regulates angiogenesis but also affects inflammation. In spite of intensive research, the function of PECAM-1 still awaits further elucidation. Although PECAM-1 undergoes alternative splicing, the full-length isoform is the predominant isoform in human endothelium. In contrast, the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform in mouse endothelium. Thus, PECAM-1 activity may be differentially regulated in humans and mice, with phosphorylation in humans and alternative splicing in mice. However, the significance of these different mechanisms for regulation of PECAM-1 activity remains unclear.
The role of the PECAM-1 isoform-specific function in various signalling pathways and regulation of eNOS activity remains unexplored. A lack of PECAM-1 is also associated with altered expression of other proangiogenic molecules, including eNOS and endoglin, in a tissue-specific manner. However, the details of the mechanisms involved are unknown. Recently, CD44 expression was also proposed as a component of the regulatory axis discussed in the present review, and its deficiency results in decreased levels of PECAM-1. This is reported to be mediated through the Hippo signalling pathway. However, the impact of CD44 deficiency on expression and activity of eNOS and endoglin needs further investigation.
The direct phosphorylation of endoglin by Src, more specifically in the Y612IY614 membrane proximal motif, induces its internalization and degradation with a significant impact on EC proliferation, migration and capillary morphogenesis . We have shown that PECAM-1 interacts with Src through its exon 13, which is present in all murine PECAM-1 isoforms . How the interaction of PECAM-1 with Src may impact on endoglin phosphorylation and EC function remains to be explored. Furthermore, the determination of whether expression of PECAM-1 isoforms, which differentially modulate EC migration, have an impact on the phosphorylation state of endoglin will provide additional insight into the mechanisms of PECAM-1 and endoglin interactions during angiogenesis.
Collectively the studies discussed in the present review establish an important role for PECAM-1 and its potential partners in regulation of angiogenesis and vascular function. Phosphorylation and/or alternative splicing of PECAM-1 has a specific impact on these activities. Thus, understanding the critical role of PECAM-1 in regulation of angiogenesis and elucidation of its isoform-specific functions will probably deepen our understanding of the angiogenesis process and provide novel targets for its modulation and potential therapeutic applications.
We greatly appreciate the help of Dr M. Ali Saghiri with preparation of the Figures.
This work was supported by grants R01 EY016995, R24 EY022883, P30 EY016665 and P30 CA014520 UW Paul P. Carbone Cancer Center Support Grant from the National Institutes of Health and an unrestricted departmental award from Research to Prevent Blindness. N.S. is a recipient of a research award from American Diabetes Association, 1-10-BS-160 and Retina Research Foundation. S.Y.P. was supported by a Predoctoral Award from AstraZeneca. C.M.S. was supported by a grant from the National Institutes of Health (R21EY023024) and the Retina Research Foundation/Daniel M. Albert Chair.
activin receptor-like kinase
basic fibroblast growth factor
epidermal growth factor
endothelial nitric oxide synthase
extracellular signal-regulated kinase
focal adhesion kinase
fibroblast growth factor
fluid shear stress
haemorrhagic telangiectasia type 1
intercellular adhesion molecule
inducible nitric oxide synthase
junctional adhesion molecule
mitogen-activated protein kinase
Madin–Darby canine kidney
neuronal nitric oxide synthase
nitric oxide synthase
platelet endothelial cell adhesion molecule
smooth muscle cell
transforming growth factor β
vascular cell adhesion molecule
vascular endothelial growth factor