Angiogenic potential of endothelial and tumor cells seeded on gelatin-based hydrogels in response to electrical stimulations.

Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP's production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5-fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In contrast, for MDA-MB-231 the production of MMPs on gelatin materials was lower compared to control materials. With the application of EF the levels of MMP-9 decreased but MMP-2 expression raised significantly for gelatin materials. Overall, the results showed that studied gelatin materials suppressed attachment of cancerous cells, as well as suppressed their angiogenic potential revealed by decreased VEGF and MMP production. Thus, this study approved gelatin-based hydrogels with proper elasticity characteristics and different degradation behavior as useful matrices for use in vascular tissue regeneration or in restriction of tumor growth after tumor resection.

Nanofiber-based hydrogels with extracellular matrix-based synthetic peptides for the prevention of capsular opacification.

Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.

The synthesis of a carbon-14 labeled pegylated Adnectin™ for placental transfer studies in guinea pigs.

Adnectins™ are novel fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interest. The molecular weights of adnectins are less than conventional monoclonal antibodies but larger than traditional small molecules. Until now, there has been no information on the placental transfer of adnectins. To assess placental permeability to adnectins in pregnant guinea pigs, a radiolabeled adnectin, ATI-1072, bound to polyethylene glycol through a [(14) C]Maleimide linker, was synthesized from [1,4-(14) C]Maleic acid. This publication describes the synthesis and analysis of PEG-[(14) C]Maleimide-adnectin ([(14) C]ATI-1072).

Development of a carbon-14 labeling approach to support disposition studies with a pegylated biologic.

Although it is widely accepted that one can extend the pharmacokinetic half-life of a therapeutic protein by covalent conjugation with polyethylene glycol (PEG), the disposition properties of such biologics have not yet been fully evaluated. Therefore, a novel [¹4C]-labeling method was developed that can be applied to a biologic conjugated with PEG through a maleimide-cysteine reaction. The method was used to study the tissue and tumor distribution of a PEGylated Adnectin, a recombinant protein derived from the 10th type III domain of fibronectin, in nude mice bearing human xenograft tumors. The PEGylated Adnectin contained a 40-kDa branched PEG (P40B) that was labeled with [¹4C] at the linker region between the PEG and Adnectin, without compromising cellular activity and plasma half-life in mice. After a single intravenous or intraperitoneal dose (33 mg/kg, 1.7 µCi per mouse) of [¹4C]-P40B-Adnectin, quantitative whole-body autoradiography analysis revealed that the liver had the highest uptake of the radioactivity among nontumor tissues, followed by the kidneys and lung. The muscle and brain showed the least penetration of the radioactivity among all tissues examined. In addition, the [¹4C]-P40B-EI-tandem penetrated into the tumor tissue, although the extent of accumulation was largely dependent on tumor type. Therefore, it was possible to assess the tissue distribution of a PEGylated biologic after it had been [¹4C] labeled using the novel method described herein.

Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) regulates cell cycle and the fibronectin synthesis in HaCaT cell migration.

Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) was studied with respect to its wound-healing effects on a human keratinocyte (HaCaT) cell line. Disaggregated collagen fibres were treated with 0.1M NaOH for 24h and digested with pepsin for 72h to reach maximum yield of 26.6%. The results of an in vitro wound-healing test showed that migration of HaCaT cells was 1.5-fold faster on PSC-coated plates than on untreated plates. The migration rate of sea cucumber PSC was similar to that of rat PSC, but five times higher than that of bovine gelatin. HaCaT cells grown on PSC-coated plates revealed increased fibronectin synthesis (6-fold and 3-fold compared to gelatin and rat PSC, respectively). Additionally, sea cucumber PSCs induced HaCaT cell proliferation by decreasing the G1 phase by 5% and maintaining a larger population (8%) of cells in mitosis. Collagen from Red Sea cucumber might be useful as an alternative to mammalian collagen in the nutraceutical and pharmaceutical industries.

HUVEC cell affinity evaluation and integrin-mediated mechanism study on PHSRN-modified polymer.

To investigate the role of the peptide Pro-His-Ser-Arg-Asn (PHSRN) in cell adhesion and growth, PHSRN- and Gly-Arg-Gly-Asp-Ser (GRGDS)-containing polymers (P-PN5 and P-GS5, respectively) were synthesized by modification of poly(D,L-lactide-co-beta-malic acid) (PLMA) with the corresponding peptides. The cell affinities of the modified polymers were evaluated by adhesion and proliferation experiments with human umbilical vein endothelial cells (HUVECs). The results showed that P-PN5 had comparable ability to that of P-GS5 in supporting HUVEC adhesion and growth. Furthermore, the integrin-mediated mechanism of cell-substrate interaction was investigated. The results showed that P-PN5 had similar binding affinity and binding strength towards α(5)ß(1) compared to those of P-GS5. The findings suggest that PHSRN is capable of mediating the adhesion and growth of HUVECs independently and that PHSRN-modified polymers might be used as biologically compatible materials.

Bilirubin recognition via molecularly imprinted supermacroporous cryogels.

Recent years molecular imprinting has received considerable attention as an excellent and simple approach to recognize small molecules and bioactive substances. The aim of this study is to prepare the bilirubin-imprinted supermacroporous cryogels which can be used for the adsorption of bilirubin from human plasma. N-methacryloyl-(L)-tyrosinemethylester (MAT) was chosen as the pre-organization monomer. In the first step, bilirubin was complexed with MAT and the bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) [BR-MIP] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After that, the template molecules (i.e., bilirubin) were removed from the polymeric structure using sodium carbonate and sodium hydroxide. The maximum bilirubin adsorption amount was 3.6 mg/g polymer. The relative selectivity coefficients of the BR-MIP cryogel for bilirubin/cholesterol and bilirubin/testosterone mixtures were 7.3 and 3.2 times greater than non-imprinted poly(HEMA-MAT) [NIP] cryogel, respectively. The BR-MIP cryogel could be used many times without decreasing bilirubin adsorption amount significantly. Therefore, as a reusable carrier possessing high selectivity, BR-MIP cryogel has a potential candidate as a clinical hemoperfusion material.

Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds.

An important challenge in regenerative medicine is the design of suitable bioactive scaffold materials that can act as artificial extracellular matrices. We reported previously on a family of peptide-amphiphile (PA) molecules that self-assemble into high-aspect ratio nanofibers under physiological conditions, and can display bioactive peptide epitopes along each nanofiber's periphery. One type of PA displays its epitope at a branched site using a lysine dendron, a molecular feature that improves epitope availability on the nanofiber surface. In this work, we describe the application of these branched PA (b-PA) systems as self-assembling coatings for fiber-bonded poly(glycolic acid) scaffolds. b-PAs bearing variations of the RGDS adhesion epitope from fibronectin were shown by elemental analysis to coat repeatably onto fiber scaffolds. The retention of supramolecular organization after coating on the scaffold was demonstrated through spectroscopic identification of beta-sheet structures and the close association of hydrophobic tails in a model pyrene-containing PA system. Primary human bladder smooth muscle cells demonstrated greater initial adhesion to b-PA-functionalized scaffolds than to bare scaffolds or to those coated with linear PAs. This strategy of molecular design and coating may have potential application in bladder tissue regeneration.

Design of a novel fibronectin-mimetic peptide-amphiphile for functionalized biomaterials.

The interaction of the alpha5beta1 integrin with its ligand, fibronectin, supports numerous adhesive functions and has an important role in health and disease. In recent years, there has been a considerable effort in designing fibronectin-mimetic peptides to target the integrin. However, to date, the therapeutic use of these peptides has been limited, as they cannot accurately mimic fibronectin's binding affinity for alpha5beta1. A peptide-amphiphile (PR_b) was synthesized with a peptide headgroup composed of four building blocks: a spacer; RGDSP, the primary recognition site for alpha5beta1; PHSRN, the synergy binding site; and a linker. The linker was designed to mimic two important criteria: the distance and the hydrophobicity/hydrophilicity between PHSRN and RGD in fibronectin. Human umbilical vein endothelial cells were seeded on different substrates and evaluated in terms of adhesion, spreading, specificity, cytoskeleton organization, focal adhesions, and secretion of extracellular fibronectin. This peptide was shown to perform comparably to fibronectin, indicating that a biomimetic approach can result in the design of novel peptides with therapeutic potential for biomaterial functionalization.

Functional activity of insulinoma cells (INS-1E) and pancreatic islets cultured in agarose cryogel sponges.

Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for culturing both isolated pancreatic islets and insulinoma cells (INS-1E). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply.

Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats.

Leukocytes have been investigated during the past decade for their roles in secondary tissue damage after ischemia/reperfusion injury. Peptide PRARIY, a synthetic fibronectin peptide, has shown an anti-adhesion effect in in vitro studies. Previous studies have demonstrated that anti-adhesion agents lead to reductions in apoptosis. The purpose of the present study was to determine whether the peptide PRARIY displays anti-inflammatory, anti-apoptotic, and neuroprotective effects following transient focal brain ischemia in rats. Twenty-six male Sprague-Dawley rats (300-350 g) were randomly divided into three groups: phosphate-buffered saline (PBS) controls, PRARI controls, and PRARIY treatments. The right middle cerebral artery was transiently occluded using a 4-0 nylon suture. One hour later, the occluder was withdrawn, and reperfusion was maintained for 48 h. Immediately after reperfusion, the peptides (20 mg/kg, dissolved in PBS) and the same volume of PBS were continuously infused through the right external carotid artery using an osmotic minipump for 24 h. Neurological deficits were examined at 3, 24, and 48 h after ischemia. Forty-eight hours after reperfusion, the rats were sacrificed for determining infarction size, leukocyte infiltration, and apoptosis in the ischemia area. Unexpectedly, PRARIY did not influence leukocyte infiltration. However, PRARIY-treated rats showed significantly functional outcome, reduction of infarction size, decrease of TUNEL positive cells, and increase of Bcl-2 (anti-apoptotic protein) positive cells in the ischemic areas when compared to the controls. These data indicate that the peptide PRARIY exerts its neuroprotective effects via supporting neural cell survival rather than anti-leukocyte recruitment following brain ischemia/reperfusion injury.

Can application of exogenous fibronectin enhance periodontal regeneration?

BACKGROUND: Although fibronectin (FN) is an important extracellular glycoprotein involved in periodontal wound healing, it is not clear whether the application of exogenous fibronectin (ExoFN) offers any clinical benefit. The purpose of this preliminary in vitro study was to determine the binding of FN from three different sources, viz. endogenous EDTA-plasma, endogenous serum and exogenous commercial purified human fibronectin in PBS buffer, to demineralized and non-demineralized root powder. METHOD: The binding of FN to a known quantity of mineralized and non-demineralized root powder by overnight incubation at 15 degrees C was studied by enzyme immunoassay (EIA) technique. The criteria for optimal performance of EIA procedure for the determination of FN was established. Particle size of powdered root structure was standardized using a Vibratory Sieve Shaker. RESULTS: The EDTA-plasma and the serum FN exhibited binding of (17.8 +/- 2.1 microg) and (6.5 +/- 4.5 microg), respectively, to the non-demineralized root powder. However, the binding was only significant for the EDTA-plasma FN (p < 0.01) when compared to controls. In the demineralized group there was no ascertainable binding of FN from either endogenous or exogenous sources. ExoFN in buffer exhibited no binding at all to the non-demineralized or demineralized root powder. CONCLUSION: The preliminary data suggest that additional plasma and serum factors may facilitate the binding of FN to root powder. High levels of FN in blood do not necessarily indicate that FN is available for binding to the root surface during periodontal surgery.

Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V.

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats. A group of recipient rats received i.v. injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice.

Pronounced mononuclear leukocyte (MNL) infiltration occurs in multiple organs of mice homozygous for a transforming growth factor beta 1 (TGF-beta 1) loss-of-function gene mutation [TGF-beta 1 (-/-)], followed by cachexia and eventually death. Consistent with the increased leukocyte adhesion and tissue infiltration, MNLs isolated from spleen, thymus, and peripheral blood of symptomatic TGF-beta 1 (-/-) mice, as compared to littermate controls, exhibited increased adhesion to extracellular matrix proteins and to endothelial cells in vitro. Incubation of TGF-beta 1 (-/-) MNLs with selected synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin (FN) significantly attenuated adhesion of these cells not only to FN but also to endothelial cells in vitro. Based on these observations, mice were treated with the FN peptides in an attempt to rescue them from tissue inflammation and cardiopulmonary failure. Daily injections of a combination of four synthetic FN peptides that interact with beta 1-integrins and/or cell surface proteoglycans blocked the massive infiltration of MNLs into the heart and lungs of TGF-beta 1 (-/-) mice. Peptide treatment initiated on day 8, coincident with the first evidence of increased leukocyte-endothelial cell interactions, not only blocked tissue infiltration but also moderated the lethal wasting syndrome.

Amino acids and peptides. XX. Preparation of beta-cyclododecyl aspartate and its application to synthesis of fibronectin- and laminin-related peptides.

beta-Cyclododecyl aspartate was prepared and its application to peptide synthesis was examined. Derivatives of beta-cyclododecyl aspartate are more likely to crystallize and should be useful for peptide synthesis. The cyclododecyl ester was much stable to bases than the benzyl ester and rather more stable than the cyclohexyl ester. It was removable by HF treatment and trifluoromethanesulfonic acid treatment. beta-Cyclododecyl aspartate was used for synthesis of laminin- and fibronectin-related peptides by solid phase and liquid phase methods.

Synthetic immunochemistry of glycohexapeptide analogues characteristic of oncofetal fibronectin. Solid-phase synthesis and antigenic activity.

Monoclonal antibody FDC-6, and its second-generation antibodies FDB-1 and FDB-4, are able to distinguish between fibronectin (FN) from fetal or cancer tissue (onco-FN) vs. FN from normal adult tissue and plasma (nor-FN). The epitope structure recognized by the above antibodies is the glycohexapeptide H-Val-(GalNAc-alpha)Thr-His-Pro-Gly-Tyr-OH (P2). In order to define further the specificity of the reactive site, we synthesized various glycopeptides based on the unglycosylated hexapeptide sequence (P1) and compared their reactivities with these antibodies. In continuation of our structure-activity relationship studies the (Asn3,Ala5)-glycohexapeptide analogue (P3) was synthesized by a solid-phase procedure. The [Ala(CN)3,Ala5]-glycopeptide (P4), owing to dehydration of the asparagine side chain amide during carboxyl activation of Fmoc-Asn-OH, was also isolated. Fmoc-[GalNAc(Ac)3-alpha]Thr-OH was used for incorporating the glycosylated amino acid residue. For the sake of comparison the epitope P2 and the hexapeptide sequence P1 were also synthesized. The final products were characterized by elemental and amino acid analyses, optical rotation, analytical HPLC, proton NMR and fast-atom bombardment mass spectroscopy. Synthetic analogues were applied to inhibit onco-FN specific MAbs FDB-1, FDB-4 and FDC-6 binding to immobilized onco-FN, and their activities were compared with onco-FN and nor-FN. P2 exhibited an activity similar to that of an intact molecule of onco-FN. Deglycosylation (P1) or replacement of amino acid (P3, P4) greatly reduced activity. Data clearly showed that P2 was the minimal essential structure of the epitope in onco-FN defined by MAbs FDB-1, FDB-4 and FDC-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers.

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-L-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer of L-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 micrograms/cm2 of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05-0.5 micrograms/cm2 also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.

Rabbit corneal epithelial cells adhere to two distinct heparin-binding synthetic peptides derived from fibronectin.

Fibronectin plays an important role in corneal reepithelialization during corneal wound healing. In this study, rabbit corneal epithelial (RCE) cell adhesion to fibronectin was further defined using proteolytic fragments of fibronectin and chemically synthesized peptides derived from the amino acid sequence of fibronectin. RCE cells adhere to intact fibronectin, the 75 kD fragment containing the RGDS (Arg-Gly-Asp-Ser) cell adhesion-promoting sequence, and the 33/66 kD cell adhesion promoting/heparin-binding fragments of fibronectin. The 75 kD fragment and the 33/66 kD fragments partially inhibited RCE cell adhesion to intact fibronectin, suggesting that these fragments represent distinct sites used by RCE cells to adhere to intact fibronectin. Two chemically synthesized peptides derived from the amino acid sequence of the 33/66 kD fragments of fibronectin, FN-C/H-I (YEKPGSPPREVVPRPRPGV) and FN-C/H-III (YRVRVTPKEKTGPMKE), directly promoted the adhesion of RCE cells. As further evidence that FN-C/H-I and FN-C/H-III play a role in the adhesion of RCE cells to the 33/66 kD fragments of fibronectin, we have shown that soluble FN-C/H-I and FN-C/H-III inhibited RCE cell adhesion on surfaces coated with the 33/66 kD fragments. In addition, polyclonal IgG against FN-C/H-I and FN-C/H-III partially blocked RCE cell adhesion to the 33/66 kD fragments, confirming that these sequences represent adhesion-promoting sites within these fragments. In contrast, two previously described peptides from the 33/66 kD fragments of fibronectin, which promoted the adhesion of a variety of cell types, FN-C/H-II (KNNQKSEPLIGRKKT) and CS-1 (DELPQLVTLPHPNLHG-PEILDVPST), did not support RCE cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

A cell-surface heparan sulfate proteoglycan mediates neural cell adhesion and spreading on a defined sequence from the C-terminal cell and heparin binding domain of fibronectin, FN-C/H II.

FN-C/H II is a heparin binding synthetic peptide from the C-terminal cell and heparin binding domain of fibronectin (FN) that mediates neuronal cell adhesion, spreading, and neurite outgrowth. Cellular interactions with FN-C/H II are inhibited by soluble heparin, suggesting that a cell-surface proteoglycan may mediate interactions with FN-C/H II (Haugen et al., 1990). To test this hypothesis further, heparan sulfate (HS) or chondroitin sulfate (CS) was removed from the cell surface by enzyme treatment. Heparitinase but not chondroitinase treatment of cells inhibited rat B104 neuroblastoma cell adhesion and spreading on FN-C/H II. Additionally, heparitinase treatment decreased the spreading of cells on the 33/66 kDa fragments containing the C-terminal heparin binding domain of FN. Furthermore, antibodies generated against a mouse melanoma HS proteoglycan (HSPG) inhibited B104 cell adhesion to FN-C/H II and the 33/66 kDa FN fragments. 35S-HSPG isolated from B104 cells directly bound to FN-C/H II both in solid phase assays and by affinity chromatography, but failed to bind to a control peptide from this region, CS1. The binding of 35S-HSPG was predominantly mediated by the HS and not the core protein of the HSPG. SDS-PAGE of iodinated HSPG demonstrated a single 78 kDa core protein following heparitinase digestion, which migrated at 51 kDa under nonreducing conditions. Anti-HSPG antibodies recognized the 78 kDa core protein by immunoblotting, and stained the surface of rat B104 neuroblastoma cells and cells of the primary neonatal rat nervous system. These results identify a cell-surface HSPG that likely mediates neuronal cell binding interactions with FN-C/H II.

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.