Dissecting the transcriptional program of phosphomannomutase 2-deficient cells: Lymphoblastoide B cell lines as a valuable model for congenital disorders of glycosylation studies.

Congenital disorders of glycosylation (CDG) include 150 genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences in multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein-Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies.

Influence of Regioselectively Sulfated Cellulose on in Vitro Vascularization of Biomimetic Bone Matrices.

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined.

CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

Galectin-8 enhances adhesion of multiple myeloma cells to vascular endothelium and is an adverse prognostic factor.

Multiple myeloma is characterized by abnormal infiltration of malignant plasma cells into bone marrow. Testing the hypothesis that bivalent galectin-8 (Gal-8) may influence homing of myeloma cells to vascular endothelium as a key prerequisite for infiltration, we analyzed the two Gal-8 splice variants (Gal-8S, Gal-8L). They differ in the length of their linker peptide connecting the two lectin domains. Both Gal-8 isoforms bind to cells of the myeloma lines Gal-8+ MOLP-8 and Gal-8- LP-1 in a glycan-inhibitable manner. Both Gal-8 isoforms led to enhanced adhesion of myeloma cells to vascular endothelium under dynamic shear stress conditions, Gal-8L (by more than 40-fold) even stronger than Gal-8S. Additional treatment of endothelial cells with tumour necrosis factor prior to the dynamic shear stress assay entailed an almost 100-fold enhanced adhesion of myeloma cells without addition of Gal-8 variants and a further 1.5-1.7-fold enhancement by addition of Gal-8 variants. We also found that elevated expression of Gal-8 in native multiple myeloma cells is an adverse prognostic factor for overall and event-free survival using patients' gene expression profile data of the total therapy 2 and 3 myeloma studies. Also, elevated concentrations of Gal-8 were detected (45%, 19/42 patients) in sera of multiple myeloma patients compared to those of healthy, age-matched donors. Both experimental and clinical data strongly point to the significance of Gal-8 for multiple myeloma development.

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

Human sialic acid acetyl esterase: Towards a better understanding of a puzzling enzyme.

Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4-8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O-acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

Functions and Biosynthesis of O-Acetylated Sialic Acids.

Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed.

Expression of Mucin-1 in multiple myeloma and its precursors: correlation with glycosylation and subcellular localization.

AIMS: Recent reports suggest a possible role for extracellular (MUC1N) and transmembrane (MUC1C) subunits of Mucin 1 (MUC1) in the pathogenesis of multiple myeloma (MM). Nuclear translocation of MUC1C is involved in activation of various oncogenic signalling pathways and both MUC1 subunits are potential therapeutic targets. We aimed at performing a comprehensive expression analysis of the MUC1 subunits in plasma cell dyscrasias. METHODS AND RESULTS: Immunohistochemistry with monoclonal antibodies against the MUC1N subunit (EMA and 5E10) tumour-associated glycoforms of MUC1N (5E5) and the MUC1C subunit were applied to a series of biopsies from normal controls (n = 10) and plasma cell dyscrasias (n = 121). Clonal plasma cells showed reduced MUC1N expression, and the 5E5 MUC1N epitope was expressed only in neoplastic plasma cells. Nuclear localization of MUC1C was equally frequent in all disease stages and did not differ from the control cases. Loss of both MUC1 subunits in MM (n = 12) was associated with significantly shorter overall survival and was more frequent in pretreated MM samples. CONCLUSIONS: Our findings indicate that aberrant glycosylation of MUC1 is an early event in the pathogenesis of MM. In contrast, MUC1C nuclear localization is not likely to be a driver of tumour progression.

Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA.

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.

GlycoCD: a repository for carbohydrate-related CD antigens.

SUMMARY: The open access comprehensive GlycoCD database application is for representation and retrieval of carbohydrate-related clusters of differentiation (CDs). The main objective of this database platform is to provide information about interactions of carbohydrate moieties with proteins that are important for identification of specific cell surface molecule with a focus on the integration of data from carbohydrate microarray databases. GlycoCD database comprises two sections: the carbohydrate recognition CD and glycan CD. It allows easy access through a user-friendly web interface to all carbohydrate-defined CDs and those that interact with carbohydrates along with other relevant information. AVAILABILITY: The database is freely available at http://glycosciences.de/glycocd/index.php CONTACT: r.s-albiez@dkfz.de.

Naturally occurring antibodies directed against carbohydrate tumor antigens.

Healthy persons carry within their pool of circulating antibodies immunoglobulins preferentially of IgM isotype, which are directed against a variety of tumor-associated antigens. In closer scrutiny of their nature, some of these antibodies could be defined as naturally occurring antibodies due to the germline configuration of the variable immunoglobulin region. The majority of these immunoglobulins recognize carbohydrate antigens which can be classified as oncofetal antigens. Many of these IgM antibodies present in the peripheral blood circulation can bind to tumor cells and of these a minor portion are also able to destroy tumor cells by several mechanisms, as for instance complement-mediated cytolysis or apoptosis. It was postulated that anti-carbohydrate antibodies are part of an anti-tumor immune response, while their presence in the peripheral blood of healthy donors is still waiting for a plausible explanation. It may be that recognition of defined epitopes, including carbohydrate sequences, by naturally occurring antibodies constitutes the humoral arm of an anti-tumor immune response as part of the often postulated tumor surveillance. The cytotoxic capacity of these antibodies inspired several research groups and pharmaceutical companies to design novel strategies of immunoglobulin-based anti-tumor immunotherapy.

An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells.

Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell's microenvironment resulting in an increased malignancy. Schmidt's imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis.The most active compound, (4-{[(ß-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin.In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(ß-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl ß-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin α(v)ß(3) was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site.These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis(hydroxymethyl)furan and benzoylated galactose imidate, is nontoxic and antagonizes cell physiological processes in vitro that are important for the dissemination and growth of tumor cells in vivo.

The human Cas1 protein: a sialic acid-specific O-acetyltransferase?

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.

Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes.

GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.

Transcriptional regulation of the vascular endothelial glycome by angiogenic and inflammatory signalling.

Vascular endothelial cells undergo many molecular changes during pathological processes such as inflammation and tumour development. Tumours such as malignant lymphomas affecting bone marrow are dependent on interactions with endothelial cells for (1) site-specific homing and (2) tumour-induced angiogenesis. Modifications in glycosylation are responsible for fine-tuning of distinct endothelial surface receptors. In order to gain a comprehensive insight into the regulation of the endothelial glycome, comprising genes encoding for sugar transporters (sugar s/t), glycosyltransferases (GT), glycan-degrading enzymes (GD) and lectins (GBP), we performed gene profiling analysis of the human bone marrow-derived microvascular endothelial cell line HBMEC-60 that resembles closely in its biological behaviour primary bone marrow endothelial cells. HBMEC were activated by either angiogenic VEGF or the inflammatory cytokine TNF. Approximately 48% (207 genes) of the 432 glycome genes tested were found to be expressed in HBMEC-60 cells. Inflammatory and angiogenic signals produce different profiles of up- or down-regulated glycome genes, most prominent changes were seen under TNF stimulation in terms of signal intensity and number of alterations. Stimulation by VEGF and TNF affected primarily genes encoding for glycosyltransferases and in particular those important for terminal modulation. For instance, an enhanced alpha2,6 sialylation was observed after TNF stimulation at the transcriptional and glycan expression level whereas transcription of ST3Gal1 sialylating in alpha2,3 position was enhanced after VEGF stimulation. Transcriptional analysis of the glycome gives insights into the differential regulation of glycosylation pathways and may help to understand the functional impact of endothelial glycosylation.

Involvement of alpha 1-2-fucosyltransferase I (FUT1) and surface-expressed Lewis(y) (CD174) in first endothelial cell-cell contacts during angiogenesis.

During the initiation of tumor associated angiogenesis endothelial cells migrate, adhere to extracellular matrix and form cell-cell contacts. Humoral factors of malignant cells conduct this process. We investigated whether cell surface expression of the carbohydrate blood group determinant Lewis(y) (CD174) and its precursor structure H2 (CD173) on endothelial cells is influenced by soluble factors of tumor cells. Using a bone marrow derived endothelial cell line we observed an enhanced expression of CD173/CD174 and transcription of FUT1, the key enzyme for their synthesis, after treatment with tumor necrosis factor-alpha (TNF-alpha) or conditioned supernatants of the leukemia cell line KG1a. CD173/CD174 are concentrated on pseudopodial extensions responsible for initial contacts between endothelial cells. Endothelial migration induced by TNF-alpha could be diminished by antibodies to CD174 as well as by siRNA induced downmodulation of FUT1 transcription. Endothelial FUT1-siRNA-transfectants displayed impaired in vitro angiogenesis when cultivated on extracellular matrix and in spheroid assays. In vivo upregulation of CD174 expression was observed immunocytologically in capillaries of tumor-infiltrated tissue. Together, our observations point to a tumor induced transcription of endothelial FUT1 and consequently an enhanced expression of CD174 which is involved in migration and early cell-cell contacts during tumor associated angiogenesis.

Expression of CD175 (Tn), CD175s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells.

The expression of the histo-blood group carbohydrate structures T-nouvelle (Tn, CD175), sialylated Tn (CD175s) and the Thomsen-Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal antibodies in flow cytometry, immunohistology and immunoprecipitation. Expression of sialylated CD176 was evaluated by comparative immunostaining with anti-CD176 antibodies before and after sialidase treatment. While only few cell lines expressed unmasked CD176, sialylated CD176 was present on all hematopoietic cell lines and native lymphocytes examined. CD175 and CD175s are preferentially expressed on erythroblastic leukemia cell lines. CD175s expression in these cells is consistent with the transcription of the gene encoding the key enzyme alpha2,6-sialyltransferase (hST6GalNAc1). The staining intensity was reduced after methanol pretreatment of cells, indicating that these glycans are partially expressed as constituents of glycosphingolipids. Immunoprecipitation and subsequent Western blotting revealed a series of distinct high molecular glycoproteins as carriers for these carbohydrate antigens. CD34 was identified as major carrier of CD176 by immunoprecipitation and microsequencing on a KG-1 subline enriched for CD176 expression. Incubation of several CD176-positive cell lines with anti-CD176 antibodies induced apoptosis of these cells, an effect not observed with anti-CD175/CD175s antibodies. Since the presence of naturally occurring anti-CD176 antibodies may represent a mechanism of immunosurveillance against CD176-positive tumor cells, we propose that sialylation of surface-expressed CD176--among other functions--protects against apoptosis.

Cytotoxic natural antibodies against human tumours: an option for anti-cancer immunotherapy?

Healthy individuals may contain in their peripheral blood antibodies which are able to destroy human tumour cells mediated either by complement-dependent cytotoxicity or by apoptosis. The largest proportion of these antibodies is of IgM isotype and directed against distinct tumour associated carbohydrate epitopes. Although the origin of these antibodies is not clear they seem to belong to the class of natural antibodies because they are not affinity matured and are encoded by distinct germ-line restricted gene families. It is most likely that this class of natural antibodies has in vivo an anti-tumour protective effect which may contribute to so-called tumour surveillance. On the other hand malignant tumour cells exert mechanisms to counteract such an antibody attack. These comprise soluble factors as well as cell surface expressed membrane complement regulatory proteins (mCRP). Further studies are needed to elucidate molecular mechanisms leading to either tumour destruction induced by natural antibodies or to overcome the protective strategies of the tumour against antibody attack.

Interaction of Poly(l-lysine)/Polysaccharide Complex Nanoparticles with Human Vascular Endothelial Cells.

Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.