Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix-associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet-derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C-terminal Ca2+-binding region of the protein. We also report that SPARC arrests monomeric collagen-supported smooth muscle cell proliferation in the late G1-phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin-dependent kinase inhibitors. Cyclin-dependent kinase-2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet-derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin-dependent kinase-2 activity.     

Construction of a Novel Extracellular Matrix using a New Genetically Engineered Epidermal Growth Factor Fused to IgG-Fc

The design of artificial extracellular matrices has attracted much attention in tissue engineering as well as in cell biology research. An immobilized recombinant epidermal growth factor (EGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as EGF-Fc) has been constructed. Mouse fibroblast Swiss 3T3 cells adhered both to EGF-Fc-coated and collagen-coated surfaces. Phosphorylation of EGF receptor in A431 cells was induced by immobilized EGF-Fc as well as soluble EGF. Immobilized EGF-Fc continuously activated mitogen-activated protein kinase (MAPK) in A431 cells whereas MAPK activation induced by soluble EGF decreased rapidly with time. The cytoskeleton of A431 cells adhering onto immobilized EGF-Fc was filopodia whereas that of the cells adhering onto collagen in the presence of soluble EGF was lammellipodia.     

Preparation and characterization of a VEGF-Fc fusion protein matrix for enhancing HUVEC growth

To enhance vascularization of hydrophobic implants in vivo, a VEGF-Fc fusion protein consisting of vascular endothelial growth factor (VEGF) fused to the immunoglobulin G Fc domain was prepared as an artificial extracellular matrix (ECM). VEGF-Fc was stably immobilized on a polystyrene plate due to the hydrophobicity of the Fc domain, and significantly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). Additionally, the use of VEGF-Fc as an ECM markedly promoted the proliferation of HUVECs longer than 72?h and induced the reorganization of actin filaments into larger stress fibers within these cells. The VEGF-Fc fusion protein may be a promising artificial ECM for enhancing endothelial cell growth.     

Alternagin-C, a disintegrin-like protein, induces vascular endothelial cell growth factor (VEGF) expression and endothelial cell proliferation in vitro

Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, interacts with the major collagen I receptor, the alpha(2)beta(1) integrin, inhibiting collagen binding. Here we show that ALT-C also inhibits the adhesion of a mouse fibroblast cell line (NIH-3T3) to collagen I (IC(50) 2.2 microm). In addition, when immobilized on plate wells, ALT-C supports the adhesion of this cell line as well as of human vein endothelial cell (HUVEC). ALT-C (3 microm) does not detach cells that were previously bound to collagen I. ALT-C (5 nm) induces HUVEC proliferation in vitro, and it inhibits the positive effect of vascular endothelial growth factor (VEGF) or FGF-2 on the proliferation of these cells, thus suggesting a common mechanism for these proteins. Gene expression analysis of human fibroblasts growing on ALT-C- or collagen-coated plates showed that ALT-C and collagen I induce a very similar pattern of gene expression. When compared with cells growing on plastic only, ALT-C up-regulates the expression of 45 genes including the VEGF gene and down-regulates the expression of 30 genes. Fibroblast VEGF expression was confirmed by RT-PCR and ELISA assay. Up-regulation of the VEGF gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates Akt/PKB phosphorylation, a signaling event involved in endothelial survival and angiogenesis. In conclusion, ALT-C acts as a survival factor, promoting adhesion and endothelial cell proliferation.     

Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP

Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of epidermal growth factor receptor (EGFR) signalling through the activation of a protein tyrosine phosphatase. The cytoplasmic tail of alpha(1) integrin selectively interacts with a ubiquitously expressed protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) and activates it after cell adhesion to collagen. The activation results in reduced EGFR phosphorylation after EGF stimulation. Introduction of the alpha(1) cytoplasmic domain peptide into cells induces phosphatase activation and inhibits EGF-induced cell proliferation and anchorage-independent growth of malignant cells. These data are the first demonstration of the regulation of TCPTP activity in vivo and represent a new molecular paradigm of integrin-mediated negative regulation of receptor tyrosine kinase signalling.     

Heparin-functionalized thermoresponsive surface

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.     

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate. Cell adhesion mediated by the mutagenized protein was inhibitable by a synthetic peptide Gly-Arg-Gly-Asp-Ser but not by a related peptide Gly-Arg-Gly-Glu-Ser, confirming that the inserted RGDS tetrapeptide served as a recognition signal for cell adhesion. Furthermore, the RGDS-containing protein was capable of adhering cells onto an immunoglobulin-coated surface which could not by itself support cell adhesion. Thus, the cell adhesive and immunoglobulin binding activities of the mutagenized protein appear to function coordinately. The protocol described here is essentially applicable to any protein and, therefore, provides a general principle in tailoring novel multifunctional proteins having cell adhesive activity.     

Vascular Permeability Factor/Vascular Endothelial Growth Factor Inhibits Anchorage-Disruption-Induced Apoptosis in Microvessel Endothelial Cells by Inducing Scaffold Formation

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, requiredde novoprotein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to αvβ5 and vitronectin but not by antibodies to αvβ3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFβ did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.     

Covalent layer-by-layer assembly of hyperbranched polyether and polyethyleneimine: multilayer films providing possibilities for surface functionalization and local drug delivery

A convenient and simple route to multifunctional surface coatings via the alternating covalent layer-by-layer (LBL) assembly of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO(2)) and polyethylenimine (PEI) is described. The in situ chemical reaction between HBPO-NO(2) and PEI onto aminolyzed substrates was rapid and mild. Results from ellipsometry measurements, contact angle measurements, and ATR-FTIR spectra confirmed the successful LBL assembly of the building blocks, and the surface reactivity of the multilayer films with HBPO-NO(2) as the outmost layer was demonstrated by the immobilization of an amine-functionalized fluorophore. Furthermore, a biomimetic surface was achieved by surface functionalization of the multilayer films with extracellular matrix protein collagen to promote the adhesion and growth of cells. The studies on the drug loading and in vitro release behaviors of the multilayer films demonstrated their application potentials in local delivery of hydrophilic and hydrophobic therapeutic agents.     

Intracellular signal transduction as a factor in the development of "smart" biomaterials for bone tissue engineering

Signal transduction involves studying the intracellular mechanisms that govern cellular responses to external stimuli such as hormones, cytokines, and also cell adhesion to biomaterials surfaces. Several events have been shown to be responsible for cellular adhesion and adaptation onto different surfaces. For instance, cytoskeletal rearrangements during cell adhesion require the recruitment of specific protein tyrosine kinases into focal adhesion structures that promote transient focal adhesion kinase and Src phosphorylations, initially modulating cell behavior. In addition, the phosphorylation of tyrosine (Y) residues have been generally accepted as a critical regulator of a wide range of cell-related processes, including cell proliferation, migration, differentiation, survival signalling, and energy metabolism. The understanding of the signaling involved on the mechanisms of osteoblast adhesion, proliferation, and differentiation on implant surfaces is fundamental for the successful design of novel "smart" materials, potentially decreasing the repair time, thereby allowing for faster patient rehabilitation.     

PhaP介导EGFR靶向多肽修饰的肿瘤靶向PHBHHx纳米药物递送载体的构建

聚羟基脂肪酸(PHA)颗粒表面结合蛋白Pha P具有与疏水性高分子材料表面紧密结合的能力,本研究将EGFR靶向多肽(ETP)与PhaP进行融合表达,构建了ETP-PhaP融合蛋白表达的重组工程菌Escherichia coli BL21(DE3)(pPI-ETP-P)。经对工程菌株的诱导表达及ETP-PhaP融合蛋白的纯化后,通过PhaP蛋白介导能够有效地将ETP-PhaP融合蛋白修饰于3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)纳米微球表面,构建成为具有EGFR靶向作用的药物递送载体。分别检测宫颈癌细胞系SiHa(EGFR高表达)和CaSKi(EGFR低表达)对ETP-PhaP修饰的PHBHHx纳米药物载体和未经修饰的纳米药物载体的吞噬情况。结果显示,纯化的ETP-PhaP融合蛋白能够很好地吸附于PHBHHx颗粒的表面,经ETP-PhaP融合蛋白修饰的PHBHHx纳米药物载体对EGFR高表达的宫颈癌Si Ha细胞的靶向效果强于EGFR低表达的CaSKi细胞系。这一结果表明了PhaP介导的PHBHHx纳米微球表面EGFR靶向多肽修饰具有简便、高效的优势,为疏水性纳米药物载体表面功能多肽修饰提供了一种新策略。     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

Regulation of the catalytic activity of PTP1B: roles for cell adhesion, tyrosine residue 66, and proline residues 309 and 310

The reversible phosphorylation of proteins on tyrosine residues is fundamental to a variety of intracellular signaling pathways and is controlled by the actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While much progress has been made in understanding the regulation of PTKs, there is still relatively little known concerning the regulation of PTPs. Using immune complex phosphatase assays, we demonstrated that the enzymatic activity of the nonreceptor type PTP, PTP1B, is regulated by cell adhesion. Placing primary human foreskin fibroblasts (HFFs) in suspension leads to a distinct increase in PTP1B activity, whereas the readhesion of suspended HFFs onto fibronectin or collagen I inhibited activity. To gain insight into the mechanisms involved, we analyzed recombinant forms of PTP1B mutated at potential regulatory sites. Our results indicated that tyrosine residue 66 is essential for maintaining activity at 37 degrees C. We also found that the C-terminal region of PTP1B and localization to the endoplasmic reticulum are not required for the inhibition of activity by cell adhesion. However, analysis of PA-PTP1B, in which alanines are substituted for prolines 309 and 310, revealed an important role for these residues as the catalytic activity of this mutant did not decrease following readhesion onto collagen I. Since the binding of p130cas and Src to PTP1B is dependent upon these proline residues, we assayed the regulation of PTP1B in mouse embryo fibroblasts deficient in these proteins. We found that neither p130cas nor Src is required for the inhibition of PTP1B activity by adhesion to extracellular matrix proteins. Additionally, pretreatment with cytochalasin D did not prevent the reduction of PTP1B activity when cells adhered to collagen I, indicating that cell spreading is not required for this regulation. The control of the catalytic activity of PTP1B by cell adhesion demonstrated in this study is likely to have important implications for growth factor and insulin signaling.     

Fibroblast growth factor 2-functionalized collagen matrices for skeletal muscle tissue engineering

Fibroblast growth factor 2 (FGF2) protein plays important roles in wound healing and tissue regeneration. Collagen is clinically used for wound care applications. We investigated the potential value of FGF2-functionalized collagen matrices for skeletal muscle tissue engineering. When C2C12 cells were treated with FGF2, cell adhesion increased after 3 and 5 days compared to the control (P < 0.05). Wound healing activity of FGF2 was slightly higher than the control through cell migration. Cell proliferation activity of FGF2-functionalized collagen matrices on C2C12 cells also increased. Taken together, FGF2 stimulated C2C12 myoblast growth by promoting cell adhesion, proliferation and wound healing activity after injury. The potential effect of FGF2-functionalized collagen matrices was also observed. Thus FGF2 stimulates skeletal muscle development and regeneration, thereby leading to potential utility for skeletal muscle tissue engineering.     

Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact.

A purified opioid-binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide-bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet-derived growth factor receptor (PDGF R), the colony-stimulating factor 1 receptor (CSF-1 R) and the c-kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.     

Porous poly (L-lactic acid) scaffolds are optimal substrates for internal colonization by A6 mesoangioblasts and immunocytochemical analyses

In this study, mouse mesoangioblasts were seeded onto bidimensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA), in the presence or absence of a type I collagen coating. The cells were observed under a scanning electron microscope and tested for their adhesion, survival and proliferation. Immunolocalization of heat shock protein (Hsp) 70, an abundant and ubiquitous intracellular protein in these cells, was also performed in sectioned cell-containing scaffolds under a confocal fluorescence microscope to determine if in situ analysis of intracellular constituents was feasible. The data show that PLLA films allow direct cell adhesion and represent an optimal support for cell growth, and that the internal surfaces of PLLA polymeric sponges can be colonized by mesoangioblasts, which can be submitted for in situ confocal microscopic analyses for possible monitoring of time-dependent expression of differentiation markers.     

Cell adhesion protein receptors as targets for transforming growth factor-beta action

Transforming growth factor-beta (TGF-beta) increases the incorporation of fibronectin and type I collagen into the extracellular matrix of fibroblasts and epithelial cells and enhances the attachment of thymocytes onto a fibronectin substratum. Investigation of the molecular basis for these effects showed that TGF-beta elevates specifically the expression of cell adhesion protein receptors. Treatment of cells with either form of TGF-beta, TGF-beta 1, or TGF-beta 2, increases the rate of receptor synthesis and the level of receptors on the cell surface. TGF-beta acts via two complementary mechanisms, elevation of receptor mRNA and faster kinetics of receptor beta subunit precursor to product conversion. The results show that the expression of cell adhesion receptors is susceptible to pretranslational and posttranslational regulation by factors that control cell morphology, proliferation, and differentiation such as TGF-beta.     

Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies.

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.     

Construction of a bFGF-tethered extracellular matrix using a coiled-coil helical interaction

A novel method for construction of biomaterials for tissue engineering was developed. Noncovalent associations between extracellular matrix (ECM) and growth factors were achieved by engineering recombinant versions of both proteins that included helical peptides that could form a coiled-coil structure. The helix A peptide, which is capable of forming a coiled-coil helical structure, was fused with a matrix protein that contains a cell-adhesive RGD sequence. The helix B peptide, which is also capable of forming a coiled-coil helical structure, was fused with basic fibroblast growth factor (bFGF). Each protein retained its original activity of promoting cell adhesion and cell proliferation, respectively. These recombinant proteins associated noncovalently through coiled-coil helix formation between helix A and helix B. The resulting complex combined the functions of both proteins, and this method of joining proteins with different functionalities could be used to develop biomaterials for tissue engineering.