In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: the role of RGD and PHSRN domains

Polymorphonuclear leukocytes, monocytes/macrophages, foreign body giant cells (FBGC), and lymphocytes are central in directing the host foreign body and inflammatory/immune reactions that impact material biostability, biocompatibility, and device efficacy. We employed the functional architecture of fibronectin to probe the mechanisms of protein-cell interaction in modulating leukocyte behavior. We demonstrated previously that the RGD and PHSRN domain of fibronectin and the spatial orientation between the motifs were crucial in regulating macrophage function in vitro. The current study delineates the role of RGD and PHSRN in modulating the host inflammatory response and macrophage behavior in vivo. Oligopeptides containing RGD and/or PHSRN domains were grafted onto a polyethyleneglycol-based substrate and subcutaneously cage implanted into rats. Peptide identity played a minimal role in modulating the host inflammatory response and adherent macrophage density. The RGD motif, either alone or at the terminal position with the PHSRN-containing flanking sequence, elicited a rapid macrophage fusion to form FBGCs at the early stage of implantation. Both the RGD motif and the PHSRN motif were important in mediating FBGC formation at the later implantation time. However, the PHSRN motif, specifically in the configuration of G3 RGDG6 PHSRNG, evoked a larger extent of FBGC coverage. Our results indicate the importance of RGD and PHSRN in modulating macrophage function in a time and orientation dependent fashion in vivo.     

Intracellular signaling involved in macrophage adhesion and FBGC formation as mediated by ligand-substrate interaction

Fibronectin and RGD- and/or PHSRN-containing oligopeptides were preadsorbed onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signaling kinases (namely protein tyrosine kinases, protein serine/threonine kinases, PI3-kinase, Src, and MAPK) in the adhesion of human primary blood-derived macrophages and the formation of foreign-body giant cells (FBGC) on these modified substrata was investigated. The involvement of individual intracellular signaling molecules in mediating macrophage adhesion dynamically varied with the culture time, substrate, and ligand. For example, fibronectin on TCPS or networks involved similar signaling events for macrophage adhesion; however, fibronectin and G(3)RGDG(6)PHSRNG, but not peptides with other RGD and/or PHSRN orientations, mediated similar signaling events for macrophage adhesion on TCPS but mediated different signaling events on networks. Depending on the substrate, a specific molecule (i.e., Src, protein kinase C) within the protein tyrosine kinase or protein serine/threonine kinase family was either an antagonist or agonist in mediating FBGC formation.     

Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement

The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir-Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine-Glycine-Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups--GRGDSP, and its synergy site Pro-His-Ser-Arg-Asn (PHSRN)--attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that beta1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the alphavbeta3 integrin, and looped GRGDSP preferentially engaged alphavbeta3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD-PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.     

Fibronectin and its role in granuloma formation

Fibronectin (F) is a high-molecular-weight glycoprotein widely presented on the cell surface of fibroblasts, monocytes, endothelial cells and several types of other cells. It is also available in the extracellular matrix of connective tissue and in plasma. Active biologically, F is involved in cell-to-cell and cell-to-substrate adhesion, migration and differentiation of cells, maintenance of cellular structure, wound healing, blood coagulation and opsonic function. In addition, F has a special affinity to collagen, heparin, fibrin and fibrinogen. The above properties suggest F relevance to evolution of productive inflammation and granuloma development. The studies of F presentation, distribution, localization, concentration, function and influence on cell transformation throughout different stages of granuloma evolution can be helpful in elucidation of so far obscure traits of granuloma histogenesis.     

Human monocyte adhesion onto RGD and PHSRN peptides delivered to the surface of a polycarbonate polyurethane using bioactive fluorinated surface modifiers

Fluorinated oligomers, when blended into polyurethane, have been shown to migrate to the surface and generate an interface that minimizes protein denaturation and reduces cell activation. This type of surface modification can be achieved with ppm quantities of a bioactive fluorinated surface modifier (BFSM), enabling the introduction of bioactive agents onto a surface in one manufacturing step. In the current study, two BFSMs were synthesized with covalently conjugated RGD and PHSRN peptides near the fluorine terminal groups, and were shown to be surface active in polyurethane blends. CyQuant cell enumeration, scanning electron microscopy, and cell viability assays all indicated that the bioactive (and fluorinated) substrates supported enhanced monocyte interaction. The simplicity of the surface modification technique and the demonstrated ability of the peptide BFSMs to influence cell attachment and spreading indicate the potential benefits and practical value of the BFSM technology in tailoring surfaces for biomaterial applications. This was specifically highlighted for human blood monocytes, a key cell involved in the early stages of wound healing.     

Alkylsilane-modified surfaces: inhibition of human macrophage adhesion and foreign body giant cell formation.

A homologous set of alkylsilane-modified glass surfaces with chain lengths ranging from methyl to octadecyl was prepared in order to examine the influence of alkyl surface chemistry on macrophage adhesion and foreign body giant cell (FBGC) formation. Contact angle and X-ray photoelectron spectroscopy analysis confirmed our silanation technique and indicated a consistent alkyl chain density independent of chain length. Human peripheral blood monocytes were isolated and cultured on these alkylsilane surfaces for a period of 10 days. The initial density of human monocytes was similar on all surfaces. Beyond day 0 the clean glass, methyl (DM and C1), propyl (C3), and hexyl (C6) surfaces maintained a high cell density and supported macrophage development. In contrast, long-term macrophage density was extremely low on the tetradecyl (C14) and octadecyl (C18) surfaces. When interleukin-4 was added to induce FBGC formation in vitro, the DM, C1, C3, and C6 surfaces supported high levels of macrophage fusion while clean glass strongly inhibited fusion. The C14 and C18 surfaces did not contain sufficient macrophages to support FBGC formation. Cage implant studies revealed that in vivo macrophage density and FBGC formation on clean glass and C6 surfaces was similar to in vitro data. In contrast to the monocyte culture results, the C18 cage implant samples supported significant FBGC formation, possibly as a result of different conditions within each experimental system. Radiotracer adsorption studies of eight human serum proteins identified the high concentration and tenacious hold of adsorbed von Willebrand factor as being possibly involved in the poor long-term macrophage density observed on C14 and C18.     

The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces

Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg-Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro-His-Ser-Arg-Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD-PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor-ligand interactions initiating signal transduction cascades that down-regulate ECM production.     

Fibronectin in the vitreous body--distribution and possible functional role.

The presence of fibronectin in the bovine vitreous was demonstrated by immunohistochemical procedures which showed a uniform coating of the vitreous collagen network. A fractional extraction of bovine vitreous was carried out in order to determine the distribution of fibronectin and glycosaminoglycans as related to collagen fibers. About half of total fibronectin could be extracted with aqueous buffers with increasing concentrations of KCl, part of fibronectin remained however strongly associated with the insoluble collagen network even after a final extraction with 4 M urea and 0.05 M DTT. Total extractable fibronectin was of the order of 76 micrograms per vitreous, corresponding to approximately 0.17 nM fibronectin. Total quantity of GAG-s determined as uronic acid were of the order of 2200 micrograms/vitreous corresponding approximately to 4400 micrograms disaccharide units that is to about 11 nM disaccharide units of GAG per vitreous. The persistence of fibronectin, strongly associated with the collagen fibers even after repeated KCl and urea-DTT extractions was confirmed using immuno-gold labelling of vitreous collagen fibers. Gold particle density on the collagen fibers increased with the molarity of KCl used for the extractions. These findings suggest that KCl mainly removed fiber associated components probably GAG-s, which hindered the immune recognition of fiber-bound fibronectin. The strong association of fibronectin with vitreous collagen suggested a modified model for vitreous structure taking in account the binding of fibronectin both by collagen and GAG-s.     

Introduction--prevention of adhesion formation: the journey continues.

One of the complications of abdominal surgery is the occurrence of intra-abdominal adhesions. The problems related to these adhesions have created a major impact on the health care system, thereby incurring a substantial cost to health care. In the field of gynaecology, adhesion-related problems include infertility, abdominal pain and bowel obstruction.     

Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths

Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.     

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.     

Effects of surface-coupled polyethylene oxide on human macrophage adhesion and foreign body giant cell formation in vitro.

Surface immobilized polyethylene oxide (PEO) has been shown to efficiently reduce protein adsorption and cellular adhesion, resulting in a biologically passive surface. To explore the in vitro effects of surface immobilized PEO on the human inflammatory cells, macrophages, and foreign body giant cells (FBGCs), we developed a diisocyanate-based method for coupling PEO to amine-modified glass, a surface previously shown to enhance macrophage adhesion and FBGC formation. Contact angle analysis and X-ray photoelectron spectroscopy confirmed the presence of PEO molecules bound to the surface and revealed that PEO molecular weight significantly influenced the efficiency of PEO coupling. We used a 10-day human monocyte culture protocol to demonstrate that the presence of surface coupled PEO molecules does not significantly decrease initial monocyte density or monocyte-derived macrophage density after 3 days. However, PEO-coupled surfaces significantly reduced long-term monocyte-derived macrophage density and virtually eliminated interleukin-4-induced FBGC formation observed at day 10. The cellular response to these PEO-coupled surfaces was related to the molecular weight of the PEO chains, which was varied between 200 Da and 18.5 kDa. These results suggest that an optimized PEO surface treatment may be effective in reducing inflammatory cell adhesion and possible degradation during the inflammatory response to an implanted biomedical device.     

Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays

In this paper, a method was introduced for the fabrication of vertically and spatially-controlled peptide nanostructures that enhance cell adhesion, proliferation, spreading on artificial surfaces. The RGD nanostructures with different heights were fabricated on gold surfaces by self-assembly technique through a nanoporous alumina mask composed of nanoscale-controlled pores. Pore diameter and spatial distribution were controlled by manipulating the pore widening time at a constant voltage during the mask fabrication process. Two-dimensional RGD nanodot, three-dimensional RGD nanorod, and RGD nanopillar arrays were carried out using various concentrations of RGD peptide solution, self-assembly times, and pore sizes, which were 74 nm, 63 nm, and 43 nm in diameter, respectively. The fabricated RGD nanodot, nanorod, and nanopillar arrays were utilized as a cell adhesion layer to evaluate the cell adhesion force, adhesion speed, spreading assay, and phosphorylation of cofilin protein in PC12, HeLa, and HEK293T normal cells. Among the three different nanostructures, RGD nanopillar arrays were found to be suitable for cellular attachment, spreading, and proliferation due to the proper arrangement of the RGD motif, which mimics in vivo conditions. Hence, our newly fabricated RGD nanostructured array can be successfully applied as a bio-platform for improving cellular functions and in in vitro tissue engineering.     

Intraperitoneal levonorgestrel-releasing intrauterine device following uterine perforation: the role of progestins in adhesion formation

BACKGROUND: Intrauterine contraception is a widely used, highly effective means of birth control. Uterine perforation is a serious, albeit rare, complication of intrauterine device (IUD) use. Although uterine perforation by levonorgestrel-releasing (20 micro g/day) intrauterine system (LNG-IUS) has already been reported, the peritoneal adhesion potential of this IUD is unknown. METHODS: The medical files of all patients diagnosed with an intra-peritoneal IUD between the years 1990-2002 at Hadassah Medical Center were reviewed. Histopathological study of peritoneal adhesion tissue adjacent to levonorgestrel medicated IUD was conducted in one case. RESULTS: Eight cases of dislocated IUDs were found. Four cases used LNG-IUS and four other cases used copper-IUD. Laparoscopy for IUD removal disclosed mild local peritoneal adhesions between omentum and pelvic organs in all cases. No difference was noted in the appearance of the peritoneum in the presence of either a copper-IUD or LNG-IUS. Histological examination of peritoneal tissue encasing the levonorgestrel-intrauterine system revealed loose connective tissue with aggregates of submesothelial cells with a pseudo-decidual change. Immunohistochemical staining for progesterone receptor was negative. CONCLUSIONS: The peritoneal adhesions potential of LNG-IUS is low, similar to that of the copper-bearing IUD.     

Peritoneal repair and post-surgical adhesion formation.

It was shown in 1919 that peritoneal healing differs from that of skin. When a defect is made in the parietal peritoneum the entire surface becomes epithelialized simultaneously and not gradually from the borders as in epidermalization of skin wounds. While multiplication and migration of mesothelial cells from the margin of the wound may play a small part in the regenerative process, it cannot play a major role, since new mesothelium develops in the centre of a large wound at the same time as it develops in the centre of a smaller one. Development of intraperitoneal adhesions is a dynamic process whereby surgically traumatized tissues in apposition bind through fibrin bridges which become organized by wound repair cells, often supporting a rich vascular supply as well as neuronal elements.     

The role of macrophage colony-stimulating factor in hepatic glucan-induced granuloma formation in the osteopetrosis mutant mouse defective in the production of macrophage colony-stimulating factor.

To elucidate the effects of macrophage colony-stimulating factor (M-CSF) on Kupffer cells and monocyte/macrophages in hepatic granuloma formation, we examined granulomas produced by glucan injection in the liver of osteopetrotic mice and littermates with or without M-CSF administration. In the osteopetrotic mice, monocytes were deficient in peripheral blood, and their number did not increase after glucan injection. Hepatic granulomas were formed in the osteopetrotic mice by glucan injection without a supply of blood monocytes. During this process, M-CSF-independent Kupffer cells proliferated, particularly before the granuloma formation, clustered in the hepatic sinusoid, and transformed into epithelioid cells and multinuclear giant cells. In the M-CSF-treated osteopetrotic mice, glucan injection induced an increase in the number of blood monocytes and formed hepatic granulomas at a nearly similar degree to that of littermate mice. Thus, it is concluded that neither monocytes nor M-CSF are necessary for granuloma formation. In contrast, Kupffer cells play a crucial role as granulomas develop in M-CSF-uninjected osteopetrotic mice.     

The role of the macrophage in cutaneous leishmaniasis.

The investigation of the role of the macrophage in cutaneous leishmaniasis has been prompted by observations of the clinical behaviour of the infection. In contrast to the self-healing oriental sore, chronic leishmaniasis is characterized by persistent lesions and leishmania recidiva by lesions that flare up locally long after clinical healing. In both clinical types, the parasite is thought to be maintained inside the macrophages. It will be shown that the normal macrophages of mice and guinea-pigs are parasitized by L. tropica; the parasite is not killed by the macrophages but it multiplies within these cells. Incubation of the macrophages with rabbit or human anti-Leishmania sera on the other hand, leads to the attachment of specific immunoglobulins to the macrophage cell surface and consequently to the prevention of parasitization by L. tropica under the experimental conditions. The parasite appears to be immobilized at the macrophage cell surface. Normal rabbit or human sera did not interfere with parasitization. It is postulated that the parasite specifically immobilized at the cell surface might possibly be better exposed to and affected by the immune response than intracellular parasites. Furthermore, infected parasitized macrophages contribute to the immune response by processing soluble antigens from the intracellular parasites and presenting them on their surfaces, as seen by the greater affinity (higher dilution) of anti-Leishmania antibody for the cell membrane of infected macrophages than for normal macrophages.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.