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Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott-Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.

The appearance of lipid-rich foam cells is a major feature of vulnerable atherosclerotic plaque formation. The transformation of macrophages into foam cells results from excessive uptake of cholesterol-rich particles by scavenger receptors such as CD68. We cloned a CD68-Fc immunoadhesin, a fusion protein consisting of the extracellular domain of the human CD68 and a human Fc domain, and investigated the function in vitro. Specific binding of CD68-Fc to OxLDL with an affinity of 10 nmol/L was determined by surface plasmon resonance and increased binding to lipid-rich human and ApoE(-/-) mice plaque tissue. This was confirmed both by immunohistochemical staining of CD68-Fc-treated paraffin sections from human plaques and by ELISA-based quantification of CD68-Fc binding to human atherosclerotic plaque extracts. In an in vitro model of macrophage/foam cell formation, CD68-Fc reduced foam cell formation significantly. This was caused both by interference of CD68-Fc with OxLDL uptake into macrophages and platelets and by the inhibition of platelet/OxLDL phagocytosis. Finally, expression of metalloproteinases by macrophages/foam cells was inhibited by CD68-Fc. In conclusion, CD68-Fc seems to be a promising new tool for preventing macrophage/foam cell formation. Thus, CD68-Fc might offer a novel therapeutic strategy for patients with acute coronary syndrome by modulating the generation of vulnerable plaques.

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel-Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

Single crystals of {FeHo(6)}I(12)Ho were obtained during the reaction of HoI(3) with metallic holmium and iron in a sealed tantalum container. The crystal structure consists of isolated holmium clusters encapsulating a single Fe atom, {FeHo(6)} ( symmetry). The rare earth metal atoms are surrounded by 12 edge-capping and six terminal iodide ligands that either connect the clusters to each other directly or via HoI(6) octa-hedra ( symmetry).

Background: Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell-derived factor-1 (SDF-1) regulates homing of CD34+ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34+ cells into endothelial progenitor cells., Methods and Results: Adherent platelets express substantial amounts of SDF-1 and recruit CD34+ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34+ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34+ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34+ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro., Conclusions: These findings imply that platelet-derived SDF-1 regulates adhesion of stem cells in vitro and in vivo and promotes differentiation of CD34+ cells to endothelial progenitor cells. Because tissue regeneration depends on recruitment of progenitor cells to peripheral vasculature and their subsequent differentiation, platelet-derived SDF-1 may contribute to vascular and myocardial regeneration.

Objective: Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs)., Methods and Results: The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin alphaMbeta2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C-dependent mechanism., Conclusions: Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Aims: Platelet collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial thrombosis. In this observational study, we examined the platelet surface expression of GPVI in patients with symptomatic coronary artery disease (CAD)., Methods and Results: We evaluated a consecutive cohort of 367 patients with symptomatic CAD, who underwent coronary angiography. The surface expression of platelet activation markers (GPVI, CD62P, and CD42b) was determined by flow cytometry. Patients with acute coronary syndrome (ACS) showed a significantly enhanced GPVI expression on admission when compared with patients with stable angina pectoris (SAP) (ACS: 21.4+/-9.7; SAP: 18.6+/-7.1 mean fluorescence intensity+/-SD; P=0.004). The expression of GPVI correlated with CD62P (r=0.702; P=0.001). Logistic regression analysis demonstrated that on admission, elevated platelet GPVI expression was associated with ACS, independent of markers of myocardial necrosis such as troponin and creatine kinase., Conclusion: Platelet GPVI surface expression is elevated in patients with ACS and is associated with imminent acute coronary events. The determination of the platelet-specific thrombotic marker GPVI may help to identify patients at risk before myocardial ischaemia is evident.

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.