Effects of Arthrospira platensis on Human Umbilical Vein Endothelial Cells.

Atherosclerosis is initiated by injury or damage to the vascular endothelial cell monolayer. Therefore, the early repair of the damaged vascular endothelium by a proliferation of neighbouring endothelial cells is important to prevent atherosclerosis and thrombotic events. Arthrospira platensis (AP) has been used as a dietary supplement, mainly due to its high content of vitamins, minerals, amino acids, and pigments such as chlorophylls, carotenoids, and phycocyanin, ingredients with antioxidant, anti-inflammatory, and anti-thrombotic properties. Therefore, in this prospective, placebo-controlled, data-driven, sample-size-estimated in vitro study, we tested whether an aqueous extract of AP at different concentrations (50, 100, and 200 µg/mL) had an effect on the different cellular parameters of human umbilical vein endothelial cells. Therefore, cell impedance measurement and cell proliferation were measured to investigate the monolayer formation. In addition, cell viability, integrity, and metabolism were analysed to evaluate singular cellular functions, especially the antithrombotic state. Furthermore, cell-cell and cell-substrate interactions were observed. The highest proliferation was achieved after the addition of 100 µg/mL. This was consistently confirmed by two independent optical experiments in cell cultures 48 h and 85 h after seeding and additionally by an indirect test. At this concentration, the activation or dysfunction of HUVECs was completely prevented, as confirmed by prostacyclin and interleukin-6 levels. In conclusion, in this study, AP induced a significant increase in HUVEC proliferation without inducing an inflammatory response but altered the hemostasiological balance in favour of prostacyclin over thromboxane, thereby creating an antithrombotic state. Thus, APE could be applied in the future as an accelerator of endothelial cell proliferation after, e.g., stent placement or atherosclerosis.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Modified contrast-enhanced ultrasonography with the new high-resolution examination technique of high frame rate contrast-enhanced ultrasound (HiFR-CEUS) for characterization of liver lesions: First results.

AIM: To examine to what extent the high frame rate contrast-enhanced ultrasound (HiFR) diagnostic enables the conclusive diagnosis of liver changes with suspected malignancy. MATERIAL/METHODS: Ultrasound examinations were performed by an experienced examiner using a multifrequency probe (SC6-1) on a high-end ultrasound system (Resona 7, Mindray) to clarify liver changes that were unclear on the B-scan. A bolus of 1-2.4 ml of the Sulphur hexafluoride ultrasound microbubbles contrast agent SonoVue™ (Bracco SpA, Italy) was administered with DICOM storage of CEUS examinations from the early arterial phase (5-15 s) to the late phase (5-6 min). Based on the image files stored in the PACS, an independent reading was performed regarding image quality and finding-related diagnostic significance (0 not informative/non-diagnostic to 5 excellent image quality/confident diagnosis possible). References were clinical follow-up, if possible, comparison to promptly performed computed tomography or magnetic resonance imaging, in some cases also to histopathology. RESULTS: We examined 100 patients (42 women, 58 men, from 18 years to 90 years, mean 63±13 years) with different entities of focal and diffuse liver parenchymal changes, which could be detected in all cases with sufficient image quality with CEUS and with high image quality with HiFR-CEUS. Proportionally septate cysts were found in n = 19 cases, scars after hemihepatectomy with local reduced fat in n = 5 cases, scars after microwave ablation in n = 19 cases, hemangiomas in n = 9 cases, focal nodular hyperplasia in n = 8 cases, colorectal metastases in n = 15 cases, hepatocellular carcinoma (HCC) in n = 11 cases, Osler disease in n = 8 cases. The size of lesions ranged from 5 mm to 200 mm with a mean value of 33.1±27.8 mm. Conclusive diagnoses could be made by the experienced investigator in 97/100 cases with CEUS, confirmed by reference imaging, in parts by histopathology or follow-up. The image quality for HiFR CEUS was rated with a score of 3 to 5; 62 cases were assessed with an average of good (4 points), 27 cases with very good (5 points), and in 11 cases (3 points) still satisfactory despite aggravated acoustic conditions. The specificity of HIFR-CEUS was 97%, the sensitivity 97%, the positive predictive value 94%, the negative predictive value 99% and the accuracy 97%. CONCLUSION: HIFR-CEUS has demonstrated has demonstrated an improved image quality resulting in a high diagnostic accuracy. In the hands of an experienced investigator, HiFR-CEUS allows the assessment of focal and diffuse unclear liver parenchymal changes on B-scan and dynamic assessment of microcirculation in solid and vascular changes.

Lipophilic and Hydrophilic Compounds from Arthrospira platensis and Its Effects on Tissue and Blood Cells-An Overview.

The cyanobacterium Arthrospira platensis (Spirulina platensis) is a natural source of considerable amounts of ingredients that are relevant for nutra- and pharmaceutical uses. Different hydrophilic and hydrophobic substances can be obtained by extraction from the biomass. The respective extraction techniques determine the composition of substances in the extract and thus its biological activity. In this short review, we provide an overview of the hydrophilic compounds (phenols, phycobiliproteins, polysaccharides, and vitamins) and lipophilic ingredients (chlorophylls, vitamins, fatty acids, and glycolipids) of Arthrospira platensis. The principal influences of these substances on blood and tissue cells are briefly summarized.

In Vitro Cytotoxic Activity of an Aqueous Alkali Extract of the Tinder Conk Mushroom, Fomes fomentarius (Agaricomycetes), on Murine Fibroblasts, Human Colorectal Adenocarcinoma, and Cutaneous Melanoma Cells.

Bioactive complexes of medicinal mushrooms have become attractive as complementary anticancer remedies. Our in vitro study focused on the cytotoxicity of the polyphenol-reach and beta-glucan-containing aqueous alkali extract from Fomes fomentarius fruiting bodies (FFE) using murine fibroblasts (L929), human colon adenocarcinoma cells (Caco-2), and cutaneous melanoma cells (COLO-818). Dose-dependent FFE cytotoxicity with an half maximal inhibitory concentration of 0.44 mg/mL was observed for L929 cells upon analysis of the total number of adherent cells, degree of cell viability, cell morphology, and mitochondrial metabolic activity. Cytotoxic effects on cancer cells tested using cell impedance were dependent on FFE concentration, type of cells, and their density. As a routine in vitro model for predicting human intestinal absorption, Caco-2 cells did not react on FFE, which can indirectly support its safety for the human intestinal epithelium. Melanoma cells were affected in a dose-dependent manner, even at low FFE concentrations (0.01-0.05 mg/mL). The confluent cell layer, which resembles a fully formed tumor, was much more resistant than the incompletely formed, subconfluent cell layer, simulating tumor formation. FFE applied topically could be a promising candidate to prevent melanoma development in its early stages.

Potential Effects of Nonadherent on Adherent Human Umbilical Venous Endothelial Cells in Cell Culture.

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.

Response of Endothelial Cells to Gelatin-Based Hydrogels.

The establishment of confluent endothelial cell (EC) monolayers on implanted materials has been identified as a concept to avoid thrombus formation but is a continuous challenge in cardiovascular device engineering. Here, material properties of gelatin-based hydrogels obtained by reacting gelatin with varying amounts of lysine diisocyanate ethyl ester were correlated with the functional state of hydrogel contacting venous EC (HUVEC) and HUVEC's ability to form a monolayer on these hydrogels. The density of adherent HUVEC on the softest hydrogel at 37 °C (G' = 1.02 kPa, E = 1.1 ± 0.3 kPa) was significantly lower (125 mm-1) than on the stiffer hydrogels (920 mm-1; G' = 2.515 and 5.02 kPa, E = 4.8 ± 0.8 and 10.3 ± 1.2 kPa). This was accompanied by increased matrix metalloprotease activity (9 pmol·min-2 compared to 0.6 pmol·min-2) and stress fiber formation, while cell-to-cell contacts were comparable. Likewise, release of eicosanoids (e.g., prostacyclin release of 1.7 vs 0.2 pg·mL-1·cell-1) and the pro-inflammatory cytokine MCP-1 (8 vs <1.5 pg·mL-1·cell-1) was higher on the softer than on the stiffer hydrogels. The expressions of pro-inflammatory markers COX-2, COX-1, and RAGE were slightly increased on all hydrogels on day 2 (up to 200% of the control), indicating a weak inflammation; however, the levels dropped to below the control from day 6. The study revealed that hydrogels with higher moduli approached the status of a functionally confluent HUVEC monolayer. The results indicate the promising potential especially of the discussed gelatin-based hydrogels with higher G' as biomaterials for implants foreseen for the venous system.

Phycocyanin from Arthrospira platensis as Potential Anti-Cancer Drug: Review of In Vitro and In Vivo Studies.

The application of cytostatic drugs or natural substances to inhibit cancer growth and progression is an important and evolving subject of cancer research. There has been a surge of interest in marine bioresources, particularly algae, as well as cyanobacteria and their bioactive ingredients. Dried biomass products of Arthrospira and Chlorella have been categorized as "generally recognized as safe" (GRAS) by the US Food and Drug Administration (FDA). Of particular importance is an ingredient of Arthrospira: phycocyanin, a blue-red fluorescent, water-soluble and non-toxic biliprotein pigment. It is reported to be the main active ingredient of Arthrospira and was shown to have therapeutic properties, including anti-oxidant, anti-inflammatory, immune-modulatory and anti-cancer activities. In the present review, in vitro and in vivo data on the effects of phycocyanin on various tumor cells and on cells from healthy tissues are summarized. The existing knowledge of underlying molecular mechanisms, and strategies to improve the efficiency of potential phycocyanin-based anti-cancer therapies are discussed.

Histological and SEM Assessment of Blood Stasis in Kidney Blood Vessels after Repeated Intra-Arterial Application of Radiographic Contrast Media.

BACKGROUND: After application of iodinated contrast media (CM), a pronounced deterioration of the microcirculation in skin and myocardium was reported. Clinically, the repeated application of CM, especially, led to an increase of the renal resistance index (RRI). With respect to the transiency of the RRI increase, it is reasonable to assume that the deterioration of blood flow could be due to transient blood stasis caused by reversible morphologic cell alterations due to osmotic discrepancies between CM and human blood. Therefore, the hypothesis was investigated whether CM are able to induce in vivo such blood stasis and cell deformations in the renal vasculature of well-hydrated pigs. METHODS: The in vivo study was performed as a prospective randomized examination to compare the effects of two different CM in 16 pigs (German Landrace). Pigs were randomized to receive either Iodixanol (n = 8), or Iopromide (n = 8). Each animal received 10 injections separated by 5-min intervals via the suprarenal aorta at a rate of 10 mL/s according to the usual procedure during a cardiac catheter examination. Finally, the kidneys were explanted and processed for histology (H & E staining and fibrin staining according to Weigert) as well as for scanning electron microscopy (SEM) with regards to morphologic correlates explaining the changes in the microcirculation. RESULTS: In each of the predefined four categories of vascular diameters, blood stasis were found, but clearly more often after application of Iopromide than after application of Iodixanol (p < 0.001). In addition, Iopromide induced more blood stasis in all of the examined kidney regions compared to Iodixanol (p = 0.0001). There were no obstructive events in the middle cortex following the application of Iodixanol. Except for the region around a puncture channel of a placed-in catheter probe, no fibrin was detected in Weigert's fibrin-stained samples, neither around the histologically assessed thrombi nor in vessels with blood stasis. Complementary SEM analyses revealed in a few cases only a slight generation of fibrin and thrombi and deformations, such as echinocyte and "box-like" deformations. CONCLUSIONS: According to previous in vitro studies, pathological erythrocyte deformations, such as echinocyte and box-like formation of erythrocytes, were observed also in vivo. In addition, blood stasis and/or thrombi could be detected in histological samples from explanted kidneys from young pigs after repeated in vivo administration of CM. In only a few cases, mural platelet aggregates within minimal fibrin meshes occurred only after the application of Iopromide.

Aptamer supported in vitro endothelialization of poly(ether imide) films.

Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.

Substrate-enzyme affinity-based surface modification strategy for endothelial cell-specific binding under shear stress.

Establishing an endothelial cell (EC) monolayer on top of the blood contacting surface of grafts is considered to be a promising approach for creating a hemocompatible surface. Here we utilized the high affinity interactions between the EC plasma membrane expressed enzyme called endothelin converting enzyme-1 (ECE-1) and its corresponding substrate big Endothelin-1 (bigET-1) to engineer an EC-specific binding surface. Since enzymatic cleavage of substrates require physical interaction between the enzyme and its corresponding substrate, it was hypothesized that a surface with chemically immobilized synthetic bigET-1 will preferentially attract ECs over other types of cells found in vascular system such as vascular smooth muscle cells (VSMCs). First, the expression of ECE-1 was significantly higher in ECs, and ECs processed synthetic bigET-1 to produce ET-1 in a cell number-dependent manner. Such interaction between ECs and synthetic bigET-1 was also detectible in blood. Next, vinyl-terminated self-assembled monolayers (SAMs) were established, oxidized and activated on a glass substrate as a model to immobilize synthetic bigET-1 via amide bonds. The ECs cultured on the synthetic bigET-1-immobilized surface processed larger amount of synthetic bigET-1 to produce ET-1 compared to VSMCs (102.9±5.13 vs. 9.75±0.74 pg/ml). The number of ECs bound to the synthetic bigET-1-immobilized surface during 1 h of shearing (5dyne/cm2) was approximately 3-fold higher than that of VSMCs (46.25±12.61 vs. 15.25±3.69 cells/100×HPF). EC-specific binding of synthetic bigET-1-immobilized surface over a surface modified with collagen, a common substance for cell adhesion, was also observed. The present study demonstrated that using the substrate-enzyme affinity (SEA) of cell type-specific enzyme and its corresponding substrate can be an effective method to engineer a surface preferentially binds specific type of cells. This novel strategy might open a new route toward rapid endothelialization under dynamic conditions supporting the long-term patency of cardiovascular implants.

Vascular Endothelial Cell Biology: An Update.

The vascular endothelium, a monolayer of endothelial cells (EC), constitutes the inner cellular lining of arteries, veins and capillaries and therefore is in direct contact with the components and cells of blood. The endothelium is not only a mere barrier between blood and tissues but also an endocrine organ. It actively controls the degree of vascular relaxation and constriction, and the extravasation of solutes, fluid, macromolecules and hormones, as well as that of platelets and blood cells. Through control of vascular tone, EC regulate the regional blood flow. They also direct inflammatory cells to foreign materials, areas in need of repair or defense against infections. In addition, EC are important in controlling blood fluidity, platelet adhesion and aggregation, leukocyte activation, adhesion, and transmigration. They also tightly keep the balance between coagulation and fibrinolysis and play a major role in the regulation of immune responses, inflammation and angiogenesis. To fulfill these different tasks, EC are heterogeneous and perform distinctly in the various organs and along the vascular tree. Important morphological, physiological and phenotypic differences between EC in the different parts of the arterial tree as well as between arteries and veins optimally support their specified functions in these vascular areas. This review updates the current knowledge about the morphology and function of endothelial cells, particularly their differences in different localizations around the body paying attention specifically to their different responses to physical, biochemical and environmental stimuli considering the different origins of the EC.

Epigenetics-Shedding Light on the Path Ahead for Material Sciences.

The harmonious regulation of bodily function is a necessity for healthy individuals. Looking from the viewpoint of material sciences, one can only marvel at the cellular factories, their renewal, and the overall control of messaging and control of responses. As aging progresses and/or pathologies arise, clinicians may be forced to look for replacement of organs/tissues with medical devices. Since all devices are tailored, a detailed understanding of developmental processes, including aberrant processes leading to pathologies, is crucial to provide clinicians with a suitable device. Although research in the field of epigenetics has produced effective therapeutics and diagnostic markers, our currently fragmented understanding of epigenetic processes as they relate to material development is inherently limited, with logical implications for the success of medical procedures. Here, we illustrate how material sciences for clinical applications, critically depend on all aspects of biomedical sciences, including the field of epigenetics.

Functional Glyco-Nanogels for Multivalent Interaction with Lectins.

Interactions between glycans and proteins have tremendous impact in biomolecular interactions. They are important for cell-cell interactions, proliferation and much more. Here, we emphasize the glycan-mediated interactions between pathogens and host cells. Pseudomonas aeruginosa, responsible for a huge number of nosocomial infections, is especially the focus when it comes to glycan-derivatives as pathoblockers. We present a microwave assisted protecting group free synthesis of glycomonomers based on lactose, melibiose and fucose. The monomers were polymerized in a precipitation polymerization in the presence of NiPAm to form crosslinked glyco-nanogels. The influence of reaction parameters like crosslinker type or stabilizer amount was investigated. The gels were characterized in lectin binding studies using model lectins and showed size and composition-dependent inhibition of lectin binding. Due to multivalent presentation of glycans in the gel, the inhibition was clearly stronger than with unmodified saccharides, which was compared after determination of the glycan loading. First studies with Pseudomonas aeruginosa revealed a surprising influence on the secretion of virulence factors. Functional glycogels may be in the future potent alternatives or adjuvants for antibiotic treatment of infections based on glycan interactions between host and pathogen.

Effect of lipopolysaccharide on the adherence of human umbilical vein endothelial cells (HUVEC) on a natural substrate.

 Polymers are often contaminated with lipopolysaccharides also known as endotoxins. Even small amounts of endotoxins can have strong effects on endothelial cell function so that the endothelialisation of cardiovascular implants might be hampered. An open question is how endothelial cells seeded on a body foreign substrate respond to shear load after adding Lipid A (LPA), the domain, which is responsible for much of the toxicity of gram-negative bacteria, and whether morphological changes of endothelial cells occur.LPA supplementation to the culture medium in increasing concentrations (5, 25 and 50µg/ml) resulted in progressive reductions of the density of adherent HUVEC after shear load (p < 0.001). 48% of the HUVEC in control cultures (0µg/ml LPA) were still adherent after 2 hours of shearing at 6 dyne/cm2, while 80 minutes after addition of 50µg/ml LPA, 88% of the HUVEC had already detached from the substrate and after 100 minutes no more HUVEC were attached.The results demonstrate that endotoxins are of extreme importance for the behavior of HUVEC and that in vivo pathologies can be increasingly simulated in vitro.

Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4.

Preclinical drug safety assessment includes in vitro studies with physiologically relevant cell cultures. As an in vitro system for hepatic toxicology testing, we have been generating cell clones of human hepatoblastoma cell line HepG2 by lentiviral transduction of phase I cytochrome P450 (CYP) enzymes. Here, we present a stable CYP2C19-overexpressing HepG2 cell clone (HepG2-2C19 C1) showing an enzyme activity of approximately 82 pmol x min-1 x mg-1 total cellular protein. The phenotypic stability over several passages of HepG2-2C19 C1 renders them to be a suitable reference cell clone for benchmarking CYP2C19 enzyme activity. In addition, we were interested to analyze acute cytotoxicity of the model drug cyclophosphamide (CPA) metabolized by HepG2-2C19 C1 and by a previously generated CYP3A4-overexpressing HepG2 cell clone. Upon 10 mM CPA exposure, we were able to detect its metabolites 4-hydroxy-cyclophosphamide and acrolein in CYP3A4- and CYP2C19-expressing cell clones, but not in parental HepG2 cell line. XTT and ATP assays showed a modest reduction of cell viability of not more than 50% with high dose (10 mM) CPA treatment. By contrast, dramatic acute cytotoxic effects of CPA were evident by the formation of nuclear γH2AX foci and by increased cell death events. These effects were paralleled by substantial decreases of cell membrane integrity as measured by the trypan blue exclusion test. Our data on CYP enzyme overexpressing HepG2 cell clones clearly show that cytotoxicity of CPA is dramatically underestimated by standard metabolic activity tests. Thus, additional tests to quantitate DNA damage formation and cell death induction might be required to realistically assess cytotoxicity of such compounds.

Endothelial cell migration, adhesion and proliferation on different polymeric substrates.

BACKGROUND: The formation of a functionally-confluent endothelial cell (EC) monolayer affords proliferation of EC, which only happens in case of appropriate migratory activity. AIM OF THE STUDY: The migratory pathway of human umbilical endothelial cells (HUVEC) was investigated on different polymeric substrates. MATERIAL AND METHODS: Surface characterization of the polymers was performed by contact angle measurements and atomic force microscopy under wet conditions. 30,000 HUVEC per well were seeded on polytetrafluoroethylene (PTFE) (θadv = 119°±2°), on low-attachment plate LAP (θadv = 28°±2°) and on polystyrene based tissue culture plates (TCP, θadv = 22°±1°). HUVEC tracks (trajectories) were recorded by time lapse microscopy and the euclidean distance (straight line between starting and end point), the total distance and the velocities of HUVEC not leaving the vision field were determined. RESULTS: On PTFE, 42 HUVEC were in the vision field directly after seeding. The mean length of single migration steps (SML) was 6.1±5.2 µm, the mean velocity (MV) 0.40±0.3 µm·min-1 and the complete length of the trajectory (LT) was 710±440 µm. On TCP 82 HUVEC were in the vision field subsequent to seeding. The LT was 840±550 µm, the SML 6.1±5.2 µm and the MV 0.44±0.3 µm·min-1. The trajectories on LAP differed significantly in respect to SML (2.4±3.9 µm, p < 0.05), the MV (0.16±0.3 µm·min-1, p < 0.05) and the LT (410±300 µm, p < 0.05), compared to PTFE and TCP. Solely on TCP a nearly confluent EC monolayer developed after three days. While on TCP diffuse signals of vinculin were found over the whole basal cell surface organizing the binding of the cells by focal adhesions, on PTFE vinculin was merely arranged at the cell rims, and on the hydrophilic material (LAP) no focal adhesions were found. CONCLUSION: The study revealed that the wettability of polymers affected not only the initial adherence but also the migration of EC, which is of importance for the proliferation and ultimately the endothelialization of polymer-based biomaterials.

Comparison of two substrate materials used as negative control in endothelialization studies: Glass versus polymeric tissue culture plate.

The endothelialization of synthetic surfaces applied as cardiovascular implant materials is an important issue to ensure the anti-thrombotic quality of a biomaterial. However, the rapid and constant development of a functionally-confluent endothelial cell monolayer is challenging. In order to investigate the compatibility of potential implant materials with endothelial cells several in vitro studies are performed. Here, glass and tissue culture plates (TCP) are often used as reference materials for in vitro pre-testing. However, a direct comparison of both substrates is lacking.Therefore, a comparison of study results is difficult, since results are often related to various reference materials. In this study, the endothelialization of glass and TCP was investigated in terms of adherence, morphology, integrity, viability and function using human umbilical vein endothelial cells (HUVEC).On both substrates an almost functionally confluent HUVEC monolayer was developed after nine days of cell seeding with clearly visible cell rims, decreased stress fiber formation and a pronounced marginal filament band. The viability of HUVEC was comparable for both substrates nine days after cell seeding with only a few dead cells. According to that, the cell membrane integrity as well as the metabolic activity showed no differences between TCP and glass. However, a significant difference was observed for the secretion of IL-6 and IL-8. The concentration of both cytokines, which are associated with migratory activity, was increased in the supernatant of HUVEC seeded on TCP. This result matches well with the slightly increased number of adherent HUVEC on TCP.In conclusion, these findings indicate that both reference materials are almost comparable and can be used equivalently as control materials in in vitro endothelialization studies.

CP39, CP75 and CP91 are major structural components of the Dictyostelium centrosome's core structure.

The acentriolar Dictyostelium centrosome is a nucleus-associated body consisting of a core structure with three plaque-like layers, which are surrounded by a microtubule-nucleating corona. The core duplicates once per cell cycle at the G2/M transition, whereby its central layer disappears and the two outer layers form the mitotic spindle poles. Through proteomic analysis of isolated centrosomes, we have identified CP39 and CP75, two essential components of the core structure. Both proteins can be assigned to the central core layer as their centrosomal presence is correlated to the disappearance and reappearance of the central core layer in the course of centrosome duplication. Both proteins contain domains with centrosome-binding activity in their N- and C-terminal halves, whereby the respective N-terminal half is required for cell cycle-dependent regulation. CP39 is capable of self-interaction and GFP-CP39 overexpression elicited supernumerary microtubule-organizing centers and pre-centrosomal cytosolic clusters. Underexpression stopped cell growth and reversed the MTOC amplification phenotype. In contrast, in case of CP75 underexpression of the protein by RNAi treatment elicited supernumerary MTOCs. In addition, CP75RNAi affects correct chromosome segregation and causes co-depletion of CP39 and CP91, another central core layer component. CP39 and CP75 interact with each other directly in a yeast two-hybrid assay. Furthermore, CP39, CP75 and CP91 mutually interact in a proximity-dependent biotin identification (BioID) assay. Our data indicate that these three proteins are all required for proper centrosome biogenesis and make up the major structural components of core structure's central layer.