Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.     

The quartz crystal microbalance as a continuous monitoring tool for the study of endothelial cell surface attachment and growth

The quartz crystal microbalance (QCM) was used to monitor endothelial cell (EC) adhesion on the gold surface of an oscillating quartz crystal contained in a QCM device. A number of parameters were investigated. First, we observed differential QCM O-ring toxicities for ECs. Second, appropriate conditions for cell culture and QCM cell environment were identified that can eliminate large-scale frequency oscillations in the measurements. These artifacts are not due to added cells but originate in the time-dependent evaporation of water. Having eliminated these artifacts, we then demonstrated that the measured steady-state crystal frequency shift, Delta f, and motional resistance shift, DeltaR, were determined by the number of firmly attached ECs requiring trypsinization from the crystal surface. Last, following steady-state attachment of ECs, the EC growth stimulation by fibroblast growth factor was monitored in a continuous fashion by measuring f and R values over a 72 h. period. We observed the Delta f values to increase in a way that reflected the increase in EC number bound to the QCM surface. Following addition of ECs to the QCM, the time-dependent increase in DeltaR can be interpreted in terms of increase by the ECs of the energy dissipation properties of the solution at the solution-gold surface interface. This effect is due to their rapid surface attachment and the elaboration of their cytoskeletal properties. These results indicate that the QCM technique can be used for the study of EC attachment and growth and suggest its potential for the real time study of per unit surface area cell mass distribution dynamics and viscoelastic properties and the cells' responses to stresses or perturbations brought about using biologically active molecules.     

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.     

Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay

A centrifugal force-based adhesion assay has been used to quantitatively examine the kinetics of formation of cell-cell contacts mediated specifically by expression of E-cadherin under the control of a glucocorticoid-inducible promoter in mouse fibroblasts. Analysis of cells expressing maximal or minimal levels of E-cadherin showed that the strength of E-cadherin-mediated adhesion developed in a single exponential step over a short time (half-maximal adhesion, 13-17 min). At 37 degrees C, adhesion strength increased rapidly in the first 20 min without an apparent lag phase. After 90 min, adhesion strength reached a plateau. Differences in final strengths of adhesion were commensurate with the level of E-cadherin expression. Strengthening of adhesion was temperature dependent. At 19 degrees C, strengthening of adhesion was delayed and subsequently developed with a slower rate compared to adhesion at 37 degrees C. At 4 degrees C, adhesion was completely inhibited. Strengthening of adhesion was absolutely dependent on a functional actin cytoskeleton since adhesion did not develop when cells were treated with cytochalasin D. Together, our current and previous (McNeill et al., 1993.J. Cell Biol. 120:1217-1226) studies indicate that the rate of initial strengthening of E-cadherin- mediated adhesion is neither dependent on the amount of E-cadherin expressed nor on long-range protein diffusion in the membrane to the adhesion site. However, initial strengthening of adhesion is dependent on temperature-sensitive cellular activities that may locally couple clusters of E-cadherin to the actin cytoskeleton.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.     

MAGI-1 is required for Rap1 activation upon cell-cell contact and for enhancement of vascular endothelial cadherin-mediated cell adhesion

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with beta-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca2+ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca2+ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.     

Cooperation of C1q receptors and integrins in C1q-mediated endothelial cell adhesion and spreading

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these responses are presumed to be mediated by the interaction of C1q with endothelial cell surface proteins, the identity of the participants is not known. In this study we examined the roles of two C1q binding proteins, cC1q-R/calreticulin and gC1q-R/p33, in C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVEC). When HDMVEC were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 microg/ml, a specific and dose-dependent adhesion and spreading was observed. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Spreading (68 +/- 12%) and to a moderate extent adhesion (47 +/- 9%) were inhibited by anti-gC1q-R mAb 60.11. Similar effects were noted with polyclonal anti-cC1q-R but not with control nonimmune IgG. The two Abs had a slight additive effect (75 +/- 13% inhibition) when mixed together in the proportion of 100 microg/ml anti-gC1q-R and 30 microg/ml anti-cC1q-R. More importantly, a 100% inhibition of spreading, but not adhesion, to C1q-coated wells was observed when HDMVEC were cultured in the presence of 30 microM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-beta(1) integrin Ab blocked both adhesion and spreading, anti-alpha(5) integrin blocked only spreading and not adhesion. Ag capture ELISA of endothelial cell membrane proteins using polyclonal anti-gC1q-R showed the presence of not only beta(1) and alpha(5) integrins but also CD44. Taken together these results suggest that endothelial cell adhesion and spreading require the cooperation of both C1qRs and beta(1) integrins and possibly other membrane-spanning molecules.     

Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface

The quartz crystal microbalance (QCM) is a simple, cost effective, high-resolution mass sensing technique, based upon the piezoelectric effect. As a methodology, the QCM evolved a solution measurement capability in largely analytical chemistry and electrochemistry applications due to its sensitive solution-surface interface measurement capability. The technique possesses a wide detection range. At the low mass end, it can detect monolayer surface coverage by small molecules or polymer films. At the upper end, it is capable of detecting much larger masses bound to the surface. These can be complex arrays of biopolymers and biomacromolecules, even whole cells. In addition, the QCM can provide information about the energy dissipating properties of the bound surface mass. Another important and unique feature of the technique is the ability to measure mass and energy dissipation properties of films while simultaneously carrying out electrochemistry on solution species or upon film systems bound to the upper electrode on the oscillating quartz crystal surface. These measurements can describe the course of electropolymerization of a film or can reveal ion or solute transport within a film during changes in the film environment or state, including the oxidation state for an electroactive film driven by the underlying surface potential. The past decade has witnessed an explosive growth in the application of the QCM technique to the study of a wide range of molecular systems at the solution-surface interface, in particular, biopolymer and biochemical systems. In this report, we start with a brief historical and technical overview. Then we discuss the application of the QCM technique to measurements involving micellar systems, self-assembling monolayers and their phase transition behavior, molecularly imprinted polymers, chemical sensors, films formed using the layer-by-layer assembly technique, and biopolymer films and point out the utility of the electrochemical capabilities of the technique to characterizing film properties, especially electroactive polymer films. We also describe the wide range of surface chemistries and attachment strategies used by investigators to bring about surface attachment and multi-layer interactions of these thin film systems. Next we review the wide range of recent applications of the technique to: studies of complex biochemical and biomimetic systems, the creation of protein and nucleic acid biosensors, studies of attached living cells and whole cell biosensor applications. Finally, we discuss future technical directions and applications of the QCM technique to areas such as drug discovery.     

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Discontinuous expression of endothelial cell adhesion molecules along initial lymphatic vessels in mesentery: the primary valve structure

BACKGROUND: Understanding lymphatic fluid uptake requires investigation of the primary valve system located at endothelial cell junctions. The objective of this study was to evaluate the expression pattern of adhesion molecules at endothelial cell junctions in an adult initial lymphatic network. METHODS AND RESULTS: Mesenteric tissues from adult male Wistar rats were labeled with antibodies against PECAM-1 and VE-cadherin. Endothelial cells along initial lymphatics and blood microvascular networks expressed both junctional molecules. In contrast to continuous junctional labeling along blood vessels, PECAM and VE-cadherin labeling patterns were discontinuous with gaps along lymphatic endothelial cell junctions. Along larger draining vessels in proximal regions of the initial lymphatic network, the majority of labeling gaps along junctions were less than 1microm. In comparison to draining vessels, terminal lymphatics exhibited a decrease in PECAM staining intensity and a decrease in endothelial cell junctional length defined by positive PECAM and VE-cadherin staining. CONCLUSION: These results suggest that primary valves responsible for unidirectional interstitial fluid uptake along initial lymphatic vessels are associated with discontinuous expression of endothelial junction molecules. This feature may render the ability to separate local membrane regions between neighboring endothelial cells.     

The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells

The mechanisms leading to focal adhesion kinase (FAK) activation remain obscure. We have investigated the role of the adaptor protein Shb in cell spreading and the regulation of FAK phosphorylation in immortalised brain endothelial (IBE) cells. Fibroblast growth factor 2 (FGF-2) stimulation lead to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding (PTB) domain of Shb. IBE cells overexpressing wild-type or R522K Shb (with an inactive Src homology 2 (SH2) domain) displayed increased FAK phosphorylation as well as enhanced spreading when seeded on collagen. FGF-2-induced tyrosine phosphorylation of Shb was dependent upon Src activity but independent of FAK activation. The use of Rat-1 fibroblasts overexpressing a temperature sensitive v-Src (tsLA29) confirmed that active Src enhanced Shb phosphorylation. The data indicate that Shb binds directly to FAK and regulates its phosphorylation leading to enhanced cell spreading in a Src-dependent manner.     

Impairment of dendritic cell functionality and steady-state number in obese mice

There is a finely tuned interplay between immune and neuroendocrine systems. Metabolic disturbances like obesity will have serious consequences on immunity both at the cellular and at the cytokine expression levels. Our in vivo results confirm the immune deficiency of ob/ob mice, leptin deficient and massively obese, characterized by a reduced Ag-specific T cell proliferation after keyhole limpet hemocyanin immunization. In this report, we show that dendritic cells (DCs), major APCs involved in T lymphocyte priming, are affected in obese mice. Both their function and their steady-state number are disturbed. We demonstrate that DCs from ob/ob mice are less potent in stimulation of allogenic T cells in vitro. This impaired functionality is not associated with altered expression of phenotypic markers but with the secretion of immunosuppressive cytokines such as TGF-beta. Moreover, we show increased in vivo steady-state number of epidermal DCs in ob/ob mice, which is not due to a migratory defect. The ob/ob mice are characterized by the absence of functional leptin, a key adipokine linking nutrition, metabolism, and immune functions. Interestingly, intradermal injection of leptin is able to restore epidermal DC number in obese mice. Thus, DCs might be directly sensitive to metabolic disturbances, providing a partial explanation of the immunodeficiency associated with obesity.     

Rho GAPs and GEFs: Controling switches in endothelial cell adhesion

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.