Intrinsic differences in the mechanisms of Tie2 binding to angiopoietins exploited by directed evolution to create an Ang2-selective ligand trap

Angiopoietins Ang1 and Ang2 are secreted ligands for the endothelial receptor tyrosine kinase Tie2 essential for vascular development and maintenance. Ang1 acts as an agonist to maintain normal vessel function, whereas Ang2 acts as a Tie2 antagonist. Ang2 is increased in macular edema, sepsis, and other conditions, in which it blocks Ang1-mediated signaling, causing vascular dysfunction and contributing to disease pathology. Therefore, Ang2 is an attractive therapeutic target. Previously, we reported a Tie2 ectodomain variant that selectively binds Ang2 and acts as soluble ligand trap to sequester Ang2; however, the mechanism of Ang2-binding selectivity is unknown. In the present study, we used directed protein evolution to enhance Ang2-binding affinity of this Tie2 ectodomain trap. We examined contributions of individual residues in the ligand-binding interface of Tie2 to Ang1 and Ang2 binding. Surprisingly, different combinations of Tie2 residues were found to bind each ligand, with hydrophobic residues binding both ligands and polar residues contributing selectively to either Ang1 or Ang2 binding. Our analysis also identified a single Tie2 residue, His168, with a pivotal role in both Ang1 and Ang2 binding, enabling competition between binding ligands. In summary, this study reports an enhanced-affinity Ang2-selective ligand trap with potential for therapeutic development and reveals the mechanism behind its selectivity. It also provides the first analysis of contributions of individual Tie2 residues to Ang1 and Ang2 binding and identifies selectivity-determining residues that could be targeted in the future design of small molecule and other inhibitors of Ang2 for the treatment of vascular dysfunction.     

The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis

Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.     

The Angiopoietin ligands and Tie receptors: potential diagnostic biomarkers of vascular disease

Abstract

The Angiopoietin-1 (Angpt1)/Tie2 signaling pathway is important in regulating vascular function. Angpt1-induced Tie2 activation promotes vascular endothelial cell survival and reduces vascular leakage. Angiopoietin-2 (Angpt2), a weak agonist/antagonist of Tie2, opposes and regulates Angpt1 action. The Tie family of receptor tyrosine kinases, Tie2 and Tie1, exist as either homo-or heterodimers. The molecular complex between the receptors is also crucial in controlling Angpt1 signaling; hence, the molecular balance between Angpt1:Angpt2 and Tie2:Tie1 is important in determining endothelial integrity and vascular stability. This review presents evidence of the change observed in the Angiopoietin/Tie molecules in various pathophysiological conditions and discusses the potential clinical applications of these molecules in vascular complications.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.     

Identification and characterization of small-molecule inhibitors of Tie2 kinase

Angiopoietins and Tie2 receptor were recently identified as an endothelial cell-specific ligand-receptor system that is critical for vascular development and postnatal pathologic angiogenesis by mediating vascular integrity. In this study, we identified a series of small-molecule Tie2 inhibitors, which blocked Ang1-induced Tie2 autophosphorylation and downstream signaling with an IC(50) value at 0.3 microM. Further optimization yields improved selectivity, aqueous solubility, microsomal stability and cytochrome P450 profile for one of the compounds (compound 7). Both compound 1 and compound 7 inhibit endothelial cell tube formation. Furthermore, in a rat model of Matrigel-induced choroidal neovascularization, compound 7 significantly diminished aberrant vessel growth. Our findings demonstrate a potential clinical benefit by specifically targeting Tie2-mediated angiogenic disorders.     

Molecular control of angiopoietin signalling

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.     

Microvascular rarefaction and decreased angiogenesis in rats with fetal programming of hypertension associated with exposure to a low-protein diet in utero

In hypertension, increased peripheral vascular resistance results from vascular dysfunction with or without structural changes (vessel wall remodeling and/or microvascular rarefaction). Humans with lower birth weight exhibit evidence of vascular dysfunction. The current studies were undertaken to investigate whether in utero programming of hypertension is associated with in vivo altered response and/or abnormal vascular structure. Offspring of Wistar dams fed a normal (CTRL) or low (LP)-protein diet during gestation were studied. Mean arterial blood pressure response to ANG II was significantly increased, and depressor response to sodium nitroprusside (SNP) infusions significantly decreased in male LP adult offspring relative to CTRL. No arterial remodeling was observed in male LP compared with CTRL offspring. Capillary and arteriolar density was significantly decreased in striated muscles from LP offspring at 7 and 28 days of life but was not different in late fetal life [day 21 of gestation (E21)]. Angiogenic potential of aortic rings from LP newborn (day of birth, P0) was significantly decreased. Striated muscle expressions (Western blots) of ANG II AT(1) receptor subtype, endothelial nitric oxide synthase, angiopoietin 1 and 2, Tie 2 receptor, vascular endothelial growth factor and receptor, and platelet-derived growth factor C at E21 and P7 were unaltered by antenatal diet exposure. In conclusion, blood pressure responses to ANG II and SNP are altered, and microvascular structural changes prevail in this model of fetal programming of hypertension. The capillary rarefaction is absent in the fetus and appears in the neonatal period, in association with decreased angiogenic potential. The study suggests that intrauterine protein restriction increases susceptibility to postnatal factors resulting in microvascular rarefaction, which could represent a primary event in the genesis of hypertension.     

Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.     

Oligomerized Tie2 localizes to clathrin-coated pits in response to angiopoietin-1

The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic structure and alternative splicing of chicken angiopoietin-2

Angiopoietin-1 (Ang-1) prevents endothelial cell apoptosis and promotes blood vessel stability, while angiopoietin-2 (Ang-2), a natural antagonist of Ang-1, disrupts blood vessel structure and induces apoptosis. We have sequenced the chicken angiopoietin-2 gene that spans about 46 kb of DNA and is split in 9 exons by 8 introns. Alternative splicing of the gene gives rise to three different species of Ang-2 mRNAs: Ang-2A, Ang-2B, and Ang-2C. The three mRNA isoforms, also present in humans, codify for proteins with an identical fibrinogen-like C-terminal domain and a different coiled-coil N-terminal domain. Ang-2A and particularly Ang-2C are expressed in immature testis and regressed adult testis undergoing vascular remodeling, while their expression is barely detectable in quiescent adult testis. Conversely, Ang-2B is only detectable in adult testis. The new isoforms with truncated amino-terminal domains may modulate the Tie2 receptor during vascular angiogenesis and regression.     

Preferential activity of Tie2 promoter in arteriolar endothelium

The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most reliable markers of the endothelial phenotype, across organisms, organs, and developmental stages. However, endothelium is intrinsically heterogeneous in origin, composition and function, presenting an arteriolar/venular asymmetry. In this regard, the expression of Tie2 along the vascular tree, although thought to be homogenous, has not been systematically investigated. Therefore we questioned whether the activity of Tie2 promoter is uniform in the microvascular endothelium. To this end, we analyzed in situ the expression of the markers beta-galactosidase [LacZ(Tie2)] and green fluorescent protein (GFP) [GFP(Tie2)], placed under the Tie2 promoter in transgenic mice, in whole mount tissue samples, which allow the simultaneous evaluation of its relative distribution in various microvascular compartments. In the mesenteries of LacZ(Tie2) and GFP(Tie2) mice, we found that the activity of Tie2 promoter is asymmetrically distributed, being much stronger in arteries and arterioles than on the venular side of the vascular tree. This observation was replicated in the diaphragm of LacZ(Tie2) mice. The capillaries presented a mosaic pattern of Tie2 promoter activity. Stimulation of angiogenesis either by wounding, or by intraperitoneal injection of Vascular Endothelial Growth Factor (VEGF), revealed that the arteriolar/venular asymmetry is established at endothelial cellular level early during new capillary formation, even before the starting of the microvascular blood flow. In conclusion, a strong Tie2 promoter activity qualifies as a novel marker of the arteriolar phenotype in microvascular endothelium.     

Mutant Tie2 causing venous malformation signals through Shc

Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells. A missense mutation in the intracellular domain of Tie2 resulting in an arginine to tryptophan substitution causes an inherited form of vascular dysmorphogenesis, venous malformation (VM). The signalling pathways activated by mutant Tie2 and responsible for formation and maintenance of the abnormal vessels in VM are not known. In this study, we have sought to define these pathways by identifying phosphoproteins interacting with mutant Tie2 expressed in endothelial cells. We find R849W Tie2 is constitutively active in endothelium and recruits and phosphorylates a 52 kDa protein. This protein is identified as p52 ShcA. We show endothelial cells expressing VM-mutant Tie2 are protected from cell death and expression of dominant-negative ShcA inhibits the anti-apoptotic activity of the mutant receptor. Suppression of this pro-survival signalling could be a therapeutic option for inducing regression of lesional vessels.     

Lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells

The Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. The major intracellular signaling systems activated by Tie2 in response to Angiopoietin-1 (Ang1) include the Akt and Erk1/2 pathways. Here, we investigated the role of cholesterol-rich plasma membrane microdomains (lipid rafts) in Tie2 regulation. Tie2 could not be detected in the lipid raft fraction of human umbilical vein endothelial cells (HUVECs) unless they were first stimulated with Ang1. After stimulation, a minor fraction of Tie2 associated tightly with the lipid rafts. Treatment of HUVECs with the lipid raft disrupting agent methyl-β-cyclodextrin selectively inhibited Ang1-induced Akt phosphorylation, but not Erk1/2 phosphorylation. It has been reported that inhibition of FoxO activity is an important mechanism for Ang1-stimulated Tie2-mediated endothelial function. Consistent with this, we found that phosphorylation of FoxO mediated by Tie2 activation was attenuated by lipid raft disruption. Therefore, we propose that lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells, especially for the Akt pathway.     

Activated protein C enhances human keratinocyte barrier integrity via sequential activation of epidermal growth factor receptor and Tie2

Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.     

The angiopoietin:Tie 2 interaction: A potential target for future therapies in human vascular disease

Angiopoietin-1 and -2 are endogenous ligands for the vascular endothelial receptor tyrosine kinase Tie2. Signalling by angiopoietin-1 promotes vascular endothelial cell survival and the sprouting and reorganisation of blood vessels, as well as inhibiting activation of the vascular endothelial barrier to reduce leakage and leucocyte migration into tissues. Angiopoietin-2 generally has an opposing action, and is released naturally at times of vascular growth and inflammation. There is a significant body of emerging evidence that promoting the actions of angiopoietin-1 through Tie2 is of benefit in pathologies of vascular activation, such as sepsis, stroke, diabetic retinopathy and asthma. Similarly, methods to inhibit the actions of angiopoietin-2 are emerging and have been demonstrated to be of preclinical and clinical benefit in reducing tumour angiogenesis. Here the author reviews the evidence for potential benefits of modulation of the interaction of angiopoietins with Tie2, and the potential applications. Additionally, methods for delivery of the complex protein angiopoietin-1 are discussed, as well as potentially deleterious consequences of administering angiopoietin-1.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells

Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.     

Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.