Adhesion Of Different Cell Types Research Articles

Tunable Adhesion of Different Cell Types Modulated by Thermoresponsive Polymer Brush Thickness.

Tunable adhesion of different cell types on well-defined surfaces has attracted common interests in the field of biomaterial science and surface engineering. Herein, we demonstrate a new strategy for the regulation of cell adhesion by simply controlling the thickness of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) brushes via surface-initiated atom transfer radical polymerization (ATRP). The adhesion of different cell types (4T1, HEK293, H9C2, HUVEC, and L929) can be easily modulated by varying the thickness of PNIPAAm brushes from 5.9 ± 1.0 nm (PN1) to 69.0 ± 5.0 nm (PN6). The fluorescent staining of different cell types on a variety of surfaces reveals that the thickness of PNIPAAm brushes would regulate the assembly of F-actin and the expression of vinculin and fibronectin, which are essential in regulating the adherent status of cells. Moreover, the cellular morphologies revealed that the adherent cells are well-spread, and multiple pseudopod extensions and protrusions can be observed at the margin of cells. This work provides a facile strategy for regulating tunable adhesion of different cell types, which may find applications in tissue engineering and regenerative medicine.

Polyelectrolyte Coatings for Surface Modification of Medical Implants

Abstract Polyelectrolyte multi-layer (PEM) coatings are prepared by alternative deposition of single polyelectrolyte monolayers on charged surfaces using the Layer-by-Layer (LbL) dip coating procedure. These are nanometre scaled coatings which allow fulfilling of different technical or biological requirements. The build-up process is based on selfassembly and self-organization of polycations and polyanions on different substrates including complex geometrical structures and even closed volumes, forming homogeneous layer without defects. Depending on the proper selection of the applied polyelectrolytes, coatings with different stabilities can be prepared. Some of the coatings are stable and cannot be removed from the surface. Others are degradable and can be used as systems for controlled local drug delivery. Here we summarise the results of our experience in preparation of PEM coatings with different functionalities. PEM coatings can be used as controllable delivery system for siRNA polyplexes. They can be used to control the adhesion of different cell types on the surfaces and support e.g. the endothelialisation process on cardio-vascular medical devices as e.g. stents or reduce the immunological response of the tissue after implantation. We summarise results from physical characterisation of the coatings (e.g. film thickness, roughness, electrical charge and hydrophilicity) combined with in-vitro biological studies on adhesion of HUVEC cells.

Endocan Concentration in Kidney Transplant Recipients

IntroductionEndocan is a novel soluble dermatan sulfate proteoglycan derived from endothelium. It has the capacity of binding to different biologically active molecules associated with cellular signaling, adhesion and regulating proliferation, differentiation, migration, and adhesion of different cell types in health and pathology. Elevated endocan levels are connected with endothelial activation/damage, neo-angiogenesis, and inflammation or carcinogenesis. Materials and methodsThe level of serum endocan among 63 kidney transplant recipients on three immunosuppressives (calcineurin inhibitors, mycophenolate mofetil, steroids) in correlation with other markers of endothelial damage was estimated. Additionally, 22 healthy volunteers were studied. Using a cross-sectional study design, the markers of endothelial damage like endocan, von Willebrand factor (vWF), intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM); markers of inflammation high-sensitivity C-reactive protein (hsCRP) and IL-6; and marker of kidney function cystatin C were measured using commercially available assays. ResultsEndocan, vWF, IL-6, hsCRP, ICAM, and VCAM levels were significantly higher in kidney transplant recipients comparing to healthy volunteers. In kidney transplant recipients, endocan levels correlated with renal function (estimated glomerular filtration rate by Modification of Diet in Renal Disease, r = -0.24, P < .05, creatinine r = 0.26, P < .05), time after transplantation r = -0.24, P < .05, activity of aspartate aminotransferase r = -0.46, P < .001, alanine aminotransferase r = 0.34, P < .01), ICAM r = -0.53, P < .001, VCAM r = -0.34, P < .01, hsCRP r = 0.35, P < .01, IL-6 r = 0.28, P < .05, vWF r = 0.26, P < .05. In a multifactorial analysis, the predictors of endocan levels were creatinine, ICAM, and VCAM predicting 59% of variability. ConclusionEndocan concentration among kidney transplant recipients is potentially connected with endothelial damage dependent upon graft function and time after transplantation.

ESM1 mediates NGFR-induced invasion and metastasis in murine oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a highly invasive and metastatic malignancy. The nerve growth factor receptor (NGFR) has been observed to be expressed on a subset of cells in OSCC, and NGFR+ cells have greater tumor-initiating capacity in vivo. Further, inhibition of NGFR reduces tumor growth, indicating a functional role of this receptor; however, the mechanisms by which NGFR confers enhanced tumor formation are not known. Here, we used an established murine model of OSCC and gene expression array analysis to identify ESM1 as a downstream target gene of NGFR, critical for tumor invasion and metastasis. ESM1 encodes a protein called endocan, which has the property of regulating proliferation, differentiation, migration, and adhesion of different cell types. Incubation of NGFR+ murine OSCC cells with nerve growth factor resulted in increased expression of ESM1. Importantly, ESM1 overexpression conferred an enhanced migratory, invasive, and metastatic phenotype, similar to what has been correlated with NGFR expression. Conversely, shRNA knockdown of ESM1 in NGFR overexpressing OSCC cells abrogated the tumor growth kinetics and the invasive and metastatic properties associated with NGFR. Together, our data indicate that NGFR plays an important role in the pathogenesis and progression of OSCC via regulation of ESM1.

Endocan Concentration and Chronic Inflammatory Process Among Heart Transplant Recipients

BackgroundEndocan is a novel soluble dermatan sulfate proteoglycan derived from endothelium. It has the capacity of binding to different biologically active molecules associated with cellular signaling, adhesion, and regulating proliferation, differentiation, migration, and adhesion of different cell types in health and pathology. Its elevated level is connected with endothelial activation, neovascularization, and inflammation or carcinogenesis. MethodsThe level of serum endocan among 131 heart transplant recipients on 3-drug immunosuppression (calcineurin inhibitor, mycophenolate mofetil/mycophenolic acid, steroid) in correlation with other markers of endothelial damage was determined. In addition, 22 healthy volunteers were studied. In cross-sectional study, markers were measured with the use of commercially available assays of endothelial damage—endocan and von Willebrand factor (VWF)—inflammation—high-sensitivity C-reactive protein (hsCRP), interleukin (IL) 6—and kidney function—cystatin C. ResultsThe endocan, VWF, IL-6, hsCRP, and cystatin C levels were significantly higher in heart transplant recipients compared with healthy volunteers. In our cohort, endocan level was correlated with renal function (estimated glomerular filtration rate: r = −0.21; P < .05), creatinine (r = 0.21; P < .05), erythrocyte count (r = −0.24; P < .01), hemoglobin (r = −0.33; P < .01), N-terminal pro–B-type natriuretic peptide (r = 0.25; P < .01), cholesterol (r = −0.22; P < .05), LDL (r = −0.21; P < .05), New York Heart Association functional class (r = 0.21; P < .05), hsCRP (r = 0.32; P < .01), IL-6 (r = 0.31; P < .01), and VWF (r = 0.27; P < .01). In multifactorial analysis, the predictors of endocan levels were cholesterol level, cystatin C, and IL-6, predicting 54% of variability. ConclusionsEndocan concentration among heart transplant recipients is potentially connected with endothelial damage caused by subclinical inflammation resulting from hyperlipidemia.

Endocan: a novel circulating proteoglycan.

Endocan is a novel endothelium derived soluble dermatan sulfate proteoglycan. It has the property of binding to a wide range of bioactive molecules associated with cellular signaling and adhesion and thus regulating proliferation, differentiation, migration, and adhesion of different cell types in health and disease. An increase in tissue expression or serum level of endocan reflects endothelial activation and neovascularization which are prominent pathophysiological changes associated with inflammation and tumor progression. Consequently, endocan has been used as a blood-based and tissue-based biomarker for various cancers and inflammation and has shown promising results.

New peptide-based and animal-free coatings for animal cell culture in bioreactors

Background Anchorage dependent cells require an appropriate extracellular matrix for their survival, migration, proliferation, phenotyping and/or differentiation [1-3]. These cells interact with extracellular matrix proteins, primarily through integrins, which induces focal adhesion contacts assembly and activation of signalling pathways that regulate diverse cellular processes [4]. Culture supports usually include biochemical components allowing such cells to adhere and to reconstitute an extracellular environment close to that found in vivo. Currently, this artificial environment is achieved by extracellular matrix constituents deposition, adsorption or grafting; among them collagens, fibronectin, laminin, artificial lamina propria... [5]. However, such animal proteins used in cell culture may induce pro-inflammatory stress, be unstable against proteolysis or loose activity after adsorption [6,7]. Synthetic microenvironments should be more suitable for clinical purposes: (i) improved control of physicochemical and mechanical properties, (ii) limited risks of immunogenicity, (iii) increased biosafety (animal free) and (iv) facilitated scale-up [1]. In this framework, research has recently focused on synthetic peptides or peptidomimetics that can mimic the extracellular matrix. Such molecules can be immobilized as recognition motifs on the surface of culture supports with a greater stability and easier surfaces characterization [5]. Self-assembling peptide hydrogels could mimic the chemical and mechanical aspects of the natural extracellular matrix [8,9] by undergoing large deformations, as in mammalian tissues. They have an inherent biocompatibility and should be able to direct cell behaviour [10]. They also can be functionalized with various biologically active ligands constituting good candidates to a new range of smart biomaterials, able to ensure adhesion of different cell types [11-13]. The range of biomimetic peptides that direct cell adhesion and are recognized by integrins is large. Recognition sequences derived from different extracellular matrix proteins include RGD [1], which are specific to different cell lines [1,5,6]. In this context, this work aims at designing animal-free, chemically defined and industrially scalable coatings for animal cell culture, as an alternative to collagen, fibronectin or Matrigel for laboratory and industrial large scale applications. These are based on self-assembling short peptides bearing adhesion bioactive sequences like RGDderived or other adhesion sequences developed to coat polystyrene or polyethylene terephthalate surfaces. Adhesion sequences should be recognized by cells, which should favour their anchorage and spreading.

Macrophage Adhesion on Gelatin-Based Interpenetrating Networks Grafted with PEGylated RGD

Human blood-derived macrophage adhesion on interpenetrating networks (IPNs) composed of PEGylated RGD-modified gelatin and poly(ethylene glycol) diacrylate was studied. The interaction between biomaterial immobilized with biofunctional peptides such as RGD and macrophages is central in the design of tissue-engineering scaffolds. PEGylated RGD-modified gelatin was synthesized via several steps involving PEG derivations and characterized by high-performance liquid chromatography, mass spectroscopy, gel permeation chromatography, and the trinitrobenzenesulfonic acid method. IPNs containing modified or unmodified gelatin were cultured with human macrophages and monitored at 2, 24, 96, and 168 h. At each time point, IPNs containing gelatin modified with PEGylated RGD showed a comparable adherent macrophage density as tissue culture polystyrene and a significantly higher cell density than other IPN formulations containing unmodified gelatin or gelatin modified with PEGylated triglycine. Although surface-immobilized RGD can serve to mediate the adhesion of different cell types on the biomaterial surface, the interaction of RGD with immune/inflammatory cells such as macrophages should also be considered when assessing the potential host response of tissue-engineering scaffolds.

A Novel RGD-independent Cell Adhesion Pathway Mediated by Fibronectin-bound Tissue Transglutaminase Rescues Cells from Anoikis

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins.

Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 beta 11 and alpha 6 beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 beta 1 transfectants. Upon stimulation with the activating anti-integrin beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 beta 1 and alpha 6 beta 1 integrins with high avidity and also to alpha 6 beta 4 integrin.