Fibronectin alters the rate of formation and structure of the fibrin matrix

Plasma fibronectin is a vital component of the fibrin clot; however its role on clot structure is not clearly understood. The goal of this study was to examine the influence of fibronectin on the kinetics of formation, structural characteristics and composition of reconstituted fibrin clots or fibrin matrices. Fibrin matrices were formed by adding thrombin to 1, 2 or 4 mg/ml fibrinogen supplemented with 0–0.4 mg/ml fibronectin. The rate of fibrin matrix formation was then monitored by measuring light absorbance properties at different time points. Confocal microscopy of fluorescein conjugated fibrinogen was used to visualize the structural characteristics of fibrin matrices. The amount of fibronectin in fibrin matrices was determined through electrophoresis and immunoblotting of solubilized matrices. Fibronectin concentration positively correlated with the initial rate of fibrin matrix formation and with steady state light absorbance values of fibrin matrices. An increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin matrices. Electrophoresis and immunoblotting showed that fibronectin was covalently and non-covalently bound to fibrin matrices and in the form of high molecular weight multimers. The formation of fibronectin multimers was attributed to cross-linking of fibronectin by trace amounts Factor XIIIa. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate an influence of fibronectin on fibrin matrix structural characteristics. This data is important in developing therapies that destabilize fibrin clots.     

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development,characterization, and applications

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine‐chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long‐term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co‐cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow. Biotechnol. Bioeng. 2010;105: 982–991. © 2009 Wiley Periodicals, Inc.     

Engineered blood and lymphatic capillaries in 3-D VEGF-fibrin-collagen matrices with interstitial flow

In vitro endothelial cell organization into capillaries is a long standing challenge of tissue engineering. We recently showed the utility of low level interstitial flow in guiding the organization of endothelial cells through a 3-D fibrin matrix-containing covalently bound vascular endothelial growth factor (VEGF). Here this synergistic phenomenon was extended to explore the effects of matrix composition on in vitro capillary morphogenesis of human blood versus lymphatic endothelial cells (BECs and LECs). Different mixtures of fibrin and collagen were used in conjunction with constant concentrations of matrix-bound VEGF and slow interstitial flow over 10 days. Interestingly, the BECs and LECs each showed a distinct preference in terms of organization for matrix composition: LECs organized the most extensively in a fibrin-only matrix, while BEC organization was optimized in the compliant collagen-containing matrices. Furthermore, the BECs and LECs produced architecturally different structures; while BECs organized in thick, branched networks containing wide lumen, the LECs were elongated into slender, overlapping networks with fine lumen. These data demonstrate the importance of the 3-D matrix composition in facilitating and coordinating BEC and LEC capillary morphogenesis, which is important for in vitro vascularization of engineered tissues.     

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

Summary This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the α_vβ₃ integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the α_vβ₃ integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the α_vβ₃ integrin distribution.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 × 10⁶, 9 × 10⁶, or 18 × 10⁶ cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7 ± 1.2%, P < 0.05) compared to those cells in 20 ml freezing bags (46.7 ± 0.1%) or 1.5 ml cryovials (46.3 ± 2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2 ± 0.6%) than a 10 min cooling (56.0 ± 2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 × 10⁶ and 18 × 10⁶ cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.     

Regulation of in vitro capillary tube formation by anti-integrin antibodies

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti- integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.     

Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices

Summary Here, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1–3-d period to form capillary tubes. Blocking antibodies to the α2β1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Exogenous fibrin matrix precursors stimulate the temporal progress of nerve regeneration within a silicone chamber

The silicone chamber model permits the investigation of the cellular and molecular events underlying successful regeneration of the rat sciatic nerve across a 10 mm gap. When 25 l chambers are implanted prefilled with phosphate-buffered saline (PBS), it takes 5–7 days before sufficient fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap; at 1 week postimplantation, cellular migration into the matrix from the nerve stumps is just beginning. The temporal progress of regeneration might be stimulated if a fibrin matrix, conductive to cell migration, was provided to the nerve stumps at or shortly after the time of chamber implantation. To test this hypothesis, chambers were prefilled, at the time of implantation, with different preparations of homologous plasma. A solution of 90% platelet-free plasma dialyzed against PBS (DP) formed a fibrin matrix by 24 hours postimplantation that, like the naturally formed matrix, had a predominantly longitudinal orientation. The temporal progress of regeneration was stimulated in the DP-prefilled chambers; at 17 days postimplantation, the extents of Schwann cell migration and axonal elongation were significantly greater than in the control system. In contrast, prefilling chambers with either non-citrated plasma or DP + calcium resulted in the generation of a matrix within 8 minutes that was composed of randomly oriented fibrin polymers. These matrices significantly retarded the progress of regeneration.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae)

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4 × 10⁸ granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Phosphorus release from cyanobacterial blooms in Meiliang Bay of Lake Taihu, China

To investigate the relationship between cyanobacterial density and phosphorus release into a lake aquatic environment, in situ experiments with 2.5 L microcosms were conducted in Meiliang Bay, located in the northern part of Lake Taihu, China. The effects of different environmental factors on phosphorus release and the ways changes of water quality indexes are involved in phosphorus release were further examined. It was found that total dissolved phosphorus (TDP) concentration kept to low levels (around 0.488 mg L⁻¹) in the microcosm with the low cyanobacterial density (8.85 × 10⁷ cell L⁻¹) throughout the experimental period, whereas first-order kinetics of TDP release was observed in microcosms with intermediate (7.60 × 10⁸ cell L⁻¹) and high cyanobacterial density (3.65 × 10⁹ cell L⁻¹). Accordingly their TDP release rate constants were both approximately 0.8930 d⁻¹ in the latter two treatments. The dissolved inorganic phosphorus (DIP) concentrations also increased with the increase of cyanobacterial density in 4 days. However, the DIP decreased from 35.52 mg L⁻¹ on day 4 to 6.72 mg L⁻¹ on day 6 in microcosm with the high cyanobacterial density during the experiments. Temperature could remarkably improve phosphorus release, while disturbance and illumination had negative effects on it. In addition, both TDP and DIP concentrations were positively correlated with electronic conductivity, salinity and total dissolved solid, but negatively correlated to chlorophyll-a and cyanobacterial density when cyanobacterial density was more than 7.60 × 108 cell L⁻¹. Thus, more phosphorus can be released from cyanobacterial blooms at higher cyanobacterial densities in Meiliang Bay, which is also determined by high temperature. Higher dissolved phosphorus concentration in cyanobacteria-dominated lakes (regions) is mainly due to the decomposition of cyanobacteria during the outbreak of cyanobacterial bloom in Lake Taihu, especially in Meiliang Bay.     

Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices

Endothelial cell invasion is a key step in angiogenic blood vessel formation. Sphingosine-1-phosphate (S1P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S1P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S1P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and 15-fold increases in invasion were observed, respectively). The S1P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta1 integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta1 integrins regulated invasion in fibrin. Additionally, the S1P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S1P. In contrast, TIMP-1 had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S1P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 µg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair. c-met protooncogene; transepithelial resistance; Madin-Darby canine kidney cell