Polymer-free immobilization of a cyclic RGD peptide on a nitinol stent promotes integrin-dependent endothelial coverage of strut surfaces

This study examined the utility of a stabilized cyclic RGD peptide chemically modified to selectively bind to titanium-oxide for enhanced biocompatibility of self-expanding nitinol stents. Endothelial cells express integrin receptors that promote attachment to subendothelial matrix proteins. Integrin binding to arginine-glycine-aspartic acid (RGD) peptide derivatives mimic naturally occurring adherent interactions. Irreversible covalent surface coating of conventional nitinol stents with a cyclic RGD (cRGD) peptide highly specific for integrin alpha v beta 3 might foster endothelialization after stent implantation. A selective cRGD peptide was irreversibly immobilized onto titanium oxide-rich nitinol coupons or self-expanding stents. Functionality of the engrafted RGD peptide was demonstrated using in vitro endothelial bioassays. A subsequent 7-day in vivo endothelialization study was performed using cRGD-coated self-expanding nitinol stents in rabbits. cRGD peptide coating effectively promoted endothelial cell anchorage, migration, and proliferation confirmed by increased focal adhesions. Proof-of-concept studies of rabbit cRGD stent implants showed a significant increase in endothelial coverage above stent struts relative to stents coated with BSA (cRGD = 70.1 ± 21.9 vs. BSA = 49.9 ± 21.8%, p < 0.03). Immobilization of cRGD peptides on strut surfaces represents an innovative strategy to improve endothelialization, which may facilitate vascular healing after stent implantation.     

RGD-dependent vacuolation and lumen formation observed during endothelial cell morphogenesis in three-dimensional fibrin matrices involves the alpha(v)beta(3) and alpha(5)beta(1) integrins

Recent data have revealed the involvement of the alpha(v)beta(3) integrin in angiogenesis. However, few studies to date have provided a convincing role for this receptor in in vitro assays of endothelial cell morphogenesis where defined steps can be examined. Here, we present data showing that two integrins, alpha(v)beta(3) and alpha(5)beta(1), regulate human endothelial cell vacuolation and lumen formation in three-dimensional fibrin matrices. Cells resuspended in fibrin formed intracellular vacuoles that coalesced into lumenal structures. These morphogenic events were completely inhibited by the simultaneous addition of anti-alpha(v)beta(3) and anti-alpha(5) integrin antibodies. Complete blockade was also accomplished with a combination of the cyclic Arg-Gly-Asp (cRGD) peptide and anti-alpha(5) integrin antibodies. No blockade was observed with the control Arg-Gly-Glu (RGE) peptide alone or in combination with control antibodies. Finally, we were able to demonstrate regression of vacuoles and lumens several hours after the addition of cRGD peptides combined with anti-alpha(5) integrin antibodies. These effects were not observed with control peptides alone or in combination with control antibodies. We report here the novel involvement of both the alpha(v)beta(3) and alpha(5)beta(1) integrins in vacuolation and lumen formation in a fibrin matrix, implicating a role for multiple integrins in endothelial cell morphogenesis.     

IFATS collection: Combinatorial peptides identify alpha5beta1 integrin as a receptor for the matricellular protein SPARC on adipose stromal cells

The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the alpha5beta1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to alpha5beta1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on alpha5beta1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that alpha5beta1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides. Disclosure of potential conflicts of interest is found at the end of this article.     

Expression of integrin alphavbeta3 in gliomas correlates with tumor grade and is not restricted to tumor vasculature.

In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of alpha(v)beta(3) integrin caused by malignancy. The aim of our study was to assess the extent and pattern of alpha(v)beta(3) integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of alpha(v)beta(3) integrin and quantified by an imaging software. The expression of alpha(v)beta(3) was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of alpha(v)beta(3) integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the beta(3) integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of alpha(v)beta(3) integrin in gliomas and are of relevance for the inhibition of alpha(v)beta(3) integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.     

A monoclonal antibody specific for a cytochrome c T cell stimulatory peptide inhibits T cell responses and affects the way the peptide associates with antigen-presenting cells.

A monoclonal antibody (mAb) specific for the 93-104 segment of pigeon cytochrome c (cyt) was shown to block interleukin 2 production and proliferation by pigeon cyt-specific T cells in response to the pigeon cyt 81-104 peptide using either the LK35.2 B cell hybridoma or normal splenocytes as antigen-presenting cells (APC). The mAb inhibited the response to soluble peptide antigen presented by metabolically inactive paraformaldehyde-fixed APC but not the response to APC that were pre-pulsed with Ag. These results suggest that the mAb blocked the formation of peptide-major histocompatibility complex (MHC) class II molecule complexes at the cell surface but did not displace the peptide once bound to the MHC class II molecule. As determined by direct binding experiments using labeled peptide, the major means of free peptide association with live APC was fluid-phase endocytosis. No free peptide associated directly with the MHC class II molecule at the cell surface near 0 degrees C since APC pulsed with peptide on ice did not activate cyt-specific T cells. The mAb enhanced the association of the radiolabeled peptide with APC at 4 degrees C apparently by binding of the peptide-mAb complex to Fc receptors. By stripping molecules from the LK35.2 cell surface using a nonspecific protease it was shown that the peptide-mAb complexes were not internalized either at 4 degrees C or 37 degrees C. Since the mAb was found to stably bind the peptide at pH levels below that of endosomes (pH 5.5-6.2) even if the peptide-mAb complexes were taken up by fluid-phase endocytosis, it is likely that the peptide would not be able to associate with MHC class II molecules inside the APC. This mAb appears to inhibit T cell activation by blocking the formation of peptide-MHC class II molecule complexes at the cell surface and by interfering with uptake of the peptide into endosomes. Therefore, it is different from other antibodies that have been reported to block T cell receptor recognition of preformed peptide/MHC class II molecule complexes.     

The human intraepithelial lymphocyte marker HML-1 is an integrin consisting of a beta 7 subunit associated with a distinctive alpha chain.

The membrane antigen defined by the monoclonal antibody (mAb) HML-1 is abundantly expressed on, and largely restricted to, the T cells which populate the intestinal epithelium. We show that the mature form of the antigen is a heterodimer comprising a 150-kDa alpha chain and a 120-kDa beta chain. Direct sequencing of tryptic peptides cleaved from the purified beta chain identified this polypeptide with the integrin beta 7 isotype. cDNA clones coding for the beta 7 chain have recently been isolated from T cell cDNA libraries, but the beta 7 chain had not been identified at the protein level. No information is available concerning the primary structure of the HML-1 alpha chain. We show that this subunit is synthesized as a precursor form that undergoes, like several other integrin alpha subunits, a post-translational cleavage of a peptide bond. Among the 11 human integrin alpha chains previously identified, 10 have biochemical features and/or a distribution different from those of HML-1 alpha. One, VLA alpha 4 (CD49d), has a molecular mass of 150 kDa and is expressed on HML-1+ cells but is not recognized by HML-1 mAb. We conclude that HML-1 is a novel member of the integrin family made of the beta 7 chain and of an as-yet-undescribed human alpha chain characterized by the post-translational cleavage of a 10-kDa peptide. HML-1 is, thus, probably the human counterpart of the mouse antigen M290.     

Expression and functional activity of the very late activation antigen-4 molecule on human natural killer cells in different states of activation

In the present study we describe the expression and functional activity of the alpha4beta1 heterodimer molecule on human natural killer (NK) cells. Flow cytometric analyses showed that fresh and activated NK cells expressed high levels of very late activation antigen-4 (VLA-4) molecules. These cells bound to fibronectin (FN) and to its 38 000-MW proteolytic fragment through the VLA-4 integrin that was blocked with HP2/1 anti-alpha4 monoclonal antibodies (mAbs) and with the FN peptide fragment CS1. No inhibitory effects were observed in the presence of anti-alpha5 mAb, FN peptide fragment CS2 or other irrelevant mAb. Fresh NK cells were unable to aggregate, despite their expression of VLA-4, and only activated (cultured and lymphocyte-activated killer cells) NK cells showed homotypic aggregation with HP1/7 and HP2/4 anti-alpha4 mAb related to cellular activation. These results underline new evidence of how NK cells in different states of activation maintain different constitutive levels of alpha4beta1 integrin activity, and highlight the possibility of a different functional regulation by the cells bearing VLA-4, in the expression of these epitopes and their ability to interact with their ligands.     

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).     

CEACAM1 enhances invasion and migration of melanocytic and melanoma cells

Expression of the cell adhesion molecule CEACAM1 in melanomas is an independent factor for the risk of metastasis with a predictive value superior to that of tumor thickness. We have previously shown that CEACAM1 co-localizes at the tumor-stroma interface of invading melanoma masses with integrin beta(3) and that these two adhesion molecules interact via the CEACAM1 cytoplasmic domain. To address the functional consequences of CEACAM1 expression, we investigated invasion and migration of melanocytic and melanoma cells that stably express CEACAM1 using two different in vitro systems. Here, we demonstrate that CEACAM1 expression markedly enhances cell invasion and migration. The enhanced invasion and migration of CEACAM1-transfected cells was dependent on the presence of Tyr-488 within the full-length cytoplasmic CEACAM1 domain. Treatment with anti-CEACAM monoclonal antibodies blocked CEACAM1-enhanced cell invasion and cell migration in a dose-dependent manner. Furthermore, the enhanced invasion and migration of CEACAM1-transfected melanoma cells was blocked by integrin-antagonizing RGD peptides. Expression of integrin beta(3) induces the up-regulation of CEACAM1 in melanocytic MEL6 cells. These results strengthen the view that CEACAM1 and alpha(v)beta(3) integrin are functionally interconnected with respect to the invasive growth of melanomas.     

Multifunctional unimolecular micelles for cancer-targeted drug delivery and positron emission tomography imaging

A multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for cancer-targeted drug delivery and non-invasive positron emission tomography (PET) imaging in tumor-bearing mice. The hyperbranched amphiphilic block copolymer, Boltorn(?) H40-poly(L-glutamate-hydrazone-doxorubicin)-b-poly(ethylene glycol) (i.e., H40-P(LG-Hyd-DOX)-b-PEG), was conjugated with cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides (cRGD, for integrin α(v)β(3) targeting) and macrocyclic chelators (1,4,7-triazacyclononane-N, N', N'-triacetic acid [NOTA], for (64)Cu-labeling and PET imaging) (i.e., H40-P(LG-Hyd-DOX)-b-PEG-OCH(3)/cRGD/NOTA, also referred to as H40-DOX-cRGD). The anti-cancer drug, doxorubicin (DOX) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms (i.e., PLG) via a pH-labile hydrazone linkage to enable pH-controlled drug release. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. cRGD-conjugated unimolecular micelles (i.e., H40-DOX-cRGD) exhibited a much higher cellular uptake in U87MG human glioblastoma cells due to integrin α(v)β(3)-mediated endocytosis than non-targeted unimolecular micelles (i.e., H40-DOX), thereby leading to a significantly higher cytotoxicity. In U87MG tumor-bearing mice, H40-DOX-cRGD-(64)Cu also exhibited a much higher level of tumor accumulation than H40-DOX-(64)Cu, measured by non-invasive PET imaging and confirmed by biodistribution studies and ex vivo fluorescence imaging. We believe that unimolecular micelles formed by hyperbranched amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.     

Identification of regions in the Moloney murine leukemia virus SU protein that tolerate the insertion of an integrin-binding peptide

Targeting of retroviral vectors to specific cells has been attempted through engineering of the surface (SU) protein of the murine leukemia viruses (MuLVs), but in many cases this has adversely affected protein function and targeted delivery has been difficult to achieve. In this study, we have inserted a 15-mer peptide that binds specifically to the alpha(v)beta(3) integrin into the Moloney MuLV SU protein, including regions that are surface exposed in the crystal structure of the ecotropic receptor-binding domain. We have concentrated in particular on the variable regions VRA, VRB, and VRC, which are responsible for the use of distinct cellular receptors by different MuLV subtypes and therefore may be more likely to accommodate a heterologous binding moiety. Despite these considerations, only 8 of 26 insertion sites were tolerated, including two separate regions in VRA, a cluster of sites in VRC, and previously identified sites at the N-terminus of the protein and in the proline-rich region immediately downstream of the receptor-binding domain. When expressed on retroviral vector particles, all of the viable proteins retained the ability to bind to and transduce murine cells, although the VRC mutants and an insertion in VRA gave reduced binding and titer. Finally, although all of the viable chimeras could bind to alpha(v)beta(3) in a solid-phase binding assay, we were unable to demonstrate expanded tropism for alpha(v)beta(3)-expressing human cells. This study highlights the difficulty of engineering the Moloney MuLV SU protein, even when structural information is available, and provides guidelines for the insertion of peptide ligands into the SU protein.     

Biomimetic peptides that engage specific integrin-dependent signaling pathways and bind to calcium phosphate surfaces

Many important matrix proteins involved in bone remodeling contain separate domains that orient the protein on hydroxyapatite and interact with target cell receptors, respectively. We have designed two synthetic peptides that mimic the dual activities of these large, complex proteins by binding to calcium phosphate minerals and by engaging integrin-dependent signaling pathways in osteoblasts. The addition of either PGRGDS from osteopontin or PDGEA from collagen type I to the HAP-binding domain of statherin (N15 domain) did not alter its alpha-helical structure or diminish its affinity for hydroxyapatite. Immobilized N15-PGRGDS bound MC3T3-E1 osteoblasts predominantly via the alpha v beta 3 integrin and induced focal adhesion kinase (FAK) phosphorylation at comparable levels to immobilized osteopontin. Immobilized N15-PDGEA bound MC3T3-E1 osteoblasts predominantly through the alpha 2 beta 1 integrin and induced similar levels of FAK phosphorylation. Although both peptides induced FAK phosphorylation with similar time courses, only the N15-PDGEA peptide induced ERK1/2 phosphorylation, showing that these peptides are also capable of engaging integrin-specific signaling pathways. This peptide system can be used to study adhesion-dependent control of signaling in the context of the relevant biomineral surface and may also be useful in biomaterial and tissue engineering applications.     

The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes

Arginine-glycine-aspartic acid (RGD) ligand is often chemically attached to polycation vector to improve the transfection efficiency. However, the chemical reaction may reduce or even inactivate the biological activities of peptides. In order to retain the targeting ability and biological activities, the RGD peptide was noncovalently introduced into polycations as gene delivery systems. In this paper, the tripeptide sequence RGD was added to disulfide-containing polyethyleneimine (SS-PEI)/DNA binary complexes to evaluate the influence of RGD addition for the particle size, zeta potential, morphology, and transfection efficiency. GelRedtrade mark was used as a molecular probe to show the effect of RGD addition on the cellular uptake of complexes. In vitro transfection experiments showed that SS-PEI exhibited comparable transfection efficiency, but lower cytotoxicity in comparison with 25kDa PEI. The transfection efficiency of complexes with RGD in HeLa cells was reduced statistically significantly with the increasing content of RGD peptide, but that in 293T cells was not altered significantly with the increasing content of RGD peptide. The reduced transfection efficiency of SS-PEI/DNA complexes with RGD in HeLa cells was attributed to the targeted binding interactions between the surplus RGD and the alpha(nu)beta(3) and alpha(nu)beta(5) integrins in HeLa cells, which would prevent the binding between RGD in complexes and integrin receptor on the surface of cells as well as nonspecific endocytosis of SS-PEI/DNA complexes mediated by proteoglycan in HeLa cells.     

Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe

The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target alpha(v)beta3 integrin, is dual-labeled with a radiotracer, (111)indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of alpha(v)beta3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 microCi of (111)In and 5 nmol of near-infrared (NIR) dye, whole-body gamma scintigraphy and cw imaging was conducted. Image acquisition time was 15 min for the gamma scintigraphy images and 800 ms for the optical images acquired using an NIR sensitive intensified charge-coupled device. The results show that while the target-to-background ratio (TBR) of nuclear and optical imaging were similar for surface regions of interest and consistent with the origin of gamma and NIR radiation from a common targeted peptide, the signal-to-noise ratio (SNR) was significantly higher for optical than nuclear imaging. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets. While tomographic reconstructions are necessary to probe TBR, SNR, and contrast for interior tissues, this work demonstrates for the first time the direct comparison of molecular optical and planar nuclear imaging for surface and subsurface cancers.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of α chain, β chain,and antigenic peptide,with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogenous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 β chain. Co-transfection with DR α cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated β chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR α chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, β chain, and α chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated α chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.     

Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.     

Short-term effects of adhesion peptides on the responses of preosteoblasts to pBMP-9

Adhesion peptides are currently used to enhance the interactions of osteoblasts with biomaterials. However, little is known about the effects of adhesion peptides on cell responses to growth factors, especially the bone morphogenetic proteins (BMPs). We used adhesion peptides Ac-CGGNGERPRGDTYRAY-NH(2) (pRGD), derived from bone sialoprotein, and Ac-CGGDGEA-NH(2) (pDGEA), derived from collagen, which interact with alpha(v)beta(3) and alpha(2)beta(1) integrins, respectively. We analyzed the effects of pRGD- and pDGEA-coated polystyrene (PS) on the responses of murine MC3T3-E1 preosteoblasts to a peptide derived from human BMP-9 (pBMP-9) in serum-free medium. After 1h, pRGD favoured interactions with alpha(v) while pDGEA bound beta(1) integrin subunits. Adding pBMP-9 (400 ng/mL) increased the amount of alpha(v) integrin subunits in cell membranes on pRGD-coated PS, but had no effect on beta(1) integrin subunits. Only on this substratum, collagen type I mRNA was enhanced and the addition of pBMP-9 promoted the early cell differentiation, increasing their alkaline phosphatase (ALP) activity within 24 h. These cells also organized beta(1) integrin subunits at their focal adhesion points. Inhibiting alpha(2)beta(1) integrins by pDGEA pre-treatment decreased this ALP activity. It is therefore important to understand the impact of adhesion peptides on the early cell responses to growth factors in order to improve biomimetic materials.     

Pneumocystis carinii attachment increases expression of fibronectin-binding integrins on cultured lung cells.

Pneumocystis carinii is an extracellular pathogen which requires attachment to alveolar epithelial cells for growth and replication. Previous studies have demonstrated that the extracellular matrix protein fibronectin (Fn) facilitates attachment of P. carinii to lung cells. This study addresses the role of cell surface Fn receptors (integrins) as mediators of P. carinii attachment and demonstrates the effect of P. carinii attachment on integrin expression on cultured lung cells. To determine the role of Fn-binding integrins in P. carinii attachment, attachment of 51Cr-labelled P. carinii organisms to the lung epithelial cell line A549 was quantified in the presence or absence of anti-integrin antibodies. Antibodies to the alpha v and alpha 5 integrin subunits significantly inhibited P. carinii attachment, while addition of antibody to the alpha subunit of a non-Fn-binding integrin, alpha 2, did not affect P. carinii attachment. To further investigate the role of Fn-binding integrins in P. carinii attachment, the effect of P. carinii attachment on expression of the alpha v and alpha 5 integrin subunits was determined. A549 cells incubated with either P. carinii organisms or with the P. carinii major surface glycoprotein gp120 demonstrated a 3- to 10-fold increase in expression of the alpha 5 integrin subunit; however, neither P. carinii nor gp120 affected the expression of alpha v integrin. Furthermore, the effects of P. carinii on A549 cell alpha 5 integrin expression were attenuated by the addition of an anti-gp120 antibody which blocks P. carinii attachment to A549 cells. Therefore, P. carinii attachment to lung epithelial cells appears to be mediated by alpha v- and alpha 5-containing integrins expressed on the epithelial cell surface, and P. carinii attachment results in increased expression of the alpha 5 integrin subunit.     

Receptor-mediated endocytosis of particles by peripheral dendritic cells

Human peripheral dendritic cells (DCs) are antigen-presenting cells with the ability to internalize antigen and present antigen-derived peptides to T cells. Human DCs express several receptors on the surface for endocytosis and other recognition receptors that bind to microbes or microbial products, which are internalized and processed. Here, we report the use of nanometer-size zeolite particles as a tool to study receptor-mediated endocytosis by the two subsets of immature DCs, myeloid (mDC) and plasmacytoid (pDC) dendritic cells. A major difference in receptor-mediated endocytosis was observed between the two populations of peripheral DCs. The pDC population demonstrated an almost complete lack of receptor-mediated endocytosis of zeolite particles, whereas the mDC population demonstrated a clear receptor-mediated endocytosis. Fc receptors are expressed by both peripheral DC populations and lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are known ligands of the Toll-like receptor (TLR)-2 and TLR4, respectively, both TLRs expressed by human mDCs. An efficient receptor-mediated endocytosis of immunoglobulin G-, LTA-, and LPS-coated zeolite particles was observed by the mDC population and their endocytosing capacity depended strongly on the density of the ligand adsorbed onto the zeolite particles. In conclusion, an efficient receptor-mediated endocytosis was observed from the mDC population, whereas the pDCs demonstrated an almost complete lack of receptor-mediated endocytosis and nanometer-size dealuminated zeolite particles were a useful tool for studying receptor-mediated endocytosis in human peripheral DCs.     

Design and activity of multifunctional fibrils using receptor-specific small peptides

We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin αvβ3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-αv integrin antibody. Further, cell attachment to B133 was inhibited by anti-α2 and anti-β1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-αv and anti-β1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin αvβ3 and that RGD-B133 interacts with both integrin αvβ3 and integrin β1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering.