Association of alphavbeta3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

INTRODUCTION: Expression of adhesion molecules such as alphavbeta3 integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether alphavbeta3 expression correlated with the metastatic potential of human osteosarcoma cells. MATERIALS AND METHODS: We developed a series of sublines (LM2-LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6-8 weeks. We quantified alphavbeta3 integrin expression using flow cytometry. RESULTS: alphavbeta3 expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of alphavbeta3. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that alphavbeta3 was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. CONCLUSIONS: alphavbeta3 integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. Alphavbeta3 integrin may therefore be a potential new target for osteosarcoma.     

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.     

In silico analysis‐based identification of the target residue of integrin α6 for metastasis inhibition of basal‐like breast cancer

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp‐358 as an integrin α6‐specific vertebrate‐conserved residue and consequently as a potential therapeutic target. Because Asp‐358 is located on the surface of the β propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp‐358 competitively inhibits integrin α6 complex formation. We treated basal‐like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin‐regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.     

Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5.     

Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.     

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.     

Primary prostatic epithelial cell binding to human bone marrow stroma and the role of a2b1 integrin

Prostate cancer selectively metastasises to the bone. To investigate the importance of prostate epithelial cell adhesion to bone marrow cells in this process we examined the binding of human primary prostatic epithelial cells (PEC) to human bone marrow stromal cultures (BMS). We found that PEC derived from both malignant and benign tissue showed greater adhesion to BMS than to benign prostatic fibroblasts (median difference was 340% and 200% respectively), skin fibroblasts or plastic tissue culture plates. Adhesion to BMS grown from the bone marrow of patients with prostatic skeletal metastases was no different from those grown from normal bone marrow. The role of integrin molecules in these cell interactions was determined. Collagen type I and fibronectin were found to increase PEC adhesion whereas vitronectin and laminin did not. Inhibition studies demonstrated that although there was heterogeneity between samples, antibodies against the integrins a2 and b1 consistently inhibited PEC binding to BMS. This result was more marked for PEC derived from malignant tissue. However studies investigating the effects of disintegrins and anti-a3 and anti-a5 integrins indicated that for a percentage of patients these integrins and RGD (arginine, glycine, aspartamine)-dependent binding pathways were also involved. In summary, the results indicate that BMS are adherent to primary PEC derived from both malignant and benign tissue. The integrin a2b1 is a major contributor to this interaction.     

Cyclic FEE peptide increases human gamete fusion and potentiates its RGD-induced inhibition

BACKGROUND: Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide. METHODS: Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte. RESULTS: Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively. CONCLUSIONS: RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.     

Porcine adenovirus serotype 3 internalization is independent of CAR and alphavbeta3 or alphavbeta5 integrin

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus-adenovirus receptor (CAR) serves as a primary receptor and alphavbeta3 or alphavbeta5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, alphavbeta3 or alphavbeta5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-alphavbeta3 or anti-alphavbeta5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as alphavbeta3 or alphavbeta5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and alphavbeta3 or alphavbeta5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.     

High affinity interactions of Coxsackievirus A9 with integrin alphavbeta3 (CD51/61) require the CYDMKTTC sequence of beta3, but do not require the RGD sequence of the CAV-9 VP1 protein

Integrins are transmembrane molecules involved in numerous cell matrix, cell-cell adhesion phenomena and also utilised as viral receptors. These interactions with integrins are mediated by brief oligopeptide recognition sequences. The Arg-Gly-Asp sequence (RGD), is recognized by many integrins, including integrin alphavbeta3 (CD51/61). Coxsackievirus A9 (CAV-9), a human pathogen that has an Arg-Gly-Asp sequence in the VP1 capsid protein, has been known to be one of the many viruses that utilise integrin alphavbeta3 as a receptor. In order to determine important binding sites of CAV-9 on integrin alphavbeta3, we performed binding studies of CAV-9 on CHO-alphavbeta3, CHO-alphavbeta1 and CHO-alphavbeta1-3-1 mutant cell line, in the presence of function blocking mAb specific for integrin alphavbeta3 and natural ligand vitronectin. Our experiments show that the CYDMKTTC sequence (187-193 residue) of integrin beta3, which has been shown to be involved in ligand specificity, is an important binding site for CAV-9. We also report that an RGD-less Coxsackievirus A9 mutant can bind efficiently on the ligand binding site of integrin alphavbeta3. Thus documenting the capability of this RNA virus to interact with integrin alphavbeta3, without the presence of an Arg-Gly-Asp sequence.     

精氨酸-甘氨酸-天冬氨酸肽类似物与新生血管内皮靶向治疗

背景：血管内皮是缺血、血栓、炎症、水肿、氧化应激等病理损伤中的重要部位,选择特异性的内皮细胞靶点成为药物介入治疗的关键。 目的：分析和总结近年来精氨酸-甘氨酸-天冬氨酸肽及精氨酸-甘氨酸-天冬氨酸多肽类似物作为特异性的内皮细胞靶点的研究。 方法：分别以“RGD、整合素、靶向治疗”,“RGD、integrin 、targeted therapy” 为检索词,应用计算机检索Pubmed 数据库1998年1月至2011年12月有关文章。纳入有关血管新生的文献。排除与研究目的无关和内容重复者。保留42篇文献做进一步分析。 结果与结论：整合素αvβ3是内皮细胞病理损伤及血管新生时的特异靶点,参与内皮细胞的迁移、增殖、分化过程。精氨酸-甘氨酸-天冬氨酸肽及精氨酸-甘氨酸-天冬氨酸多肽类似物作为整合素和其配体相互作用的识别位点,能结合肿瘤或者病理损伤时新生血管表达增高的整合素αvβ3,介导细胞与细胞外基质及细胞之间的相互作用,可在新生血管显影、靶向药物递送、载体材料修饰中发挥重要作用。     

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.     

Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics

Integrin-mediated adhesion of anchorage-dependent cells to scaffolds is a critical component of tissue engineering. We investigated integrin expression by the human fetal osteoblastic cell line, hFOB 1.19 (hFOB), as a function of substratum surface wettability. The influence of surface wettability on bone cell phenotype was also examined. Plasma-treated quartz (PTQ) and glass (PTG) (hydrophilic, contact angles of 0 degrees), octadecyltrichlorosilane-treated quartz (STQ) and glass (STG) (hydrophobic, contact angles above about 100 degrees), and tissue culture polystyrene were used for cell culture. hFOB cells cultured on hydrophilic substrata displayed well-developed actin stress fibers relative to cells on hydrophobic substrata. Western blot analysis revealed that hFOB cells cultured on hydrophobic substrata (STQ or STG) express lower levels of alphav and beta3 integrin subunits than do cells on hydrophilic substrata (PTQ or PTG). This effect was more pronounced in cells on STQ than on STG. These variations in integrin expression were lessened by extended culture time. Double- labeled integrin/actin immunofluorescence confirmed Western blot results, that is, cells cultured on PTQ displayed distinct, large plaques of alphav and beta3 subunits and integrin alphavbeta3, as well as their colocalization with actin stress fiber ends, whereas cells on STQ did not display integrin plaques after 24 h and displayed only minimal plaque formation after 3 days. Vinculin, a focal adhesion protein that mediates binding between the integrin and actin cytoskeleton, appeared in Western blots to mimic the variations of alphav and beta3 expression with respect to surface wettability. Interestingly, real-time RT-PCR analysis showed that hFOB cultured on hydrophobic substrata, which have downregulated alphav and beta3 integrin subunits, displayed greater steady state mRNA levels of osteopontin, an extracellular matrix (ECM) protein containing the Arg-Gly-Asp (RGD) integrin recognition sequence, than did cells cultured on hydrophilic substrata. Our results imply that substratum surface wettability regulates integrin-mediated bone cell adhesion and further influences the expression of bone cell-ECM complexes.     

Ectopic bone formation in rat marrow stromal cell/titanium fiber mesh scaffold constructs: effect of initial cell phenotype

Titanium fiber mesh scaffolds have been shown to be a suitable material for culture of primary marrow stromal cells in an effort to create tissue engineered constructs for bone tissue replacement. In native bone tissue, these cells are known to attach to extracellular matrix molecules via integrin receptors for specific peptide sequences, and these attachments can be a source of cell signaling, affecting cell behaviors such as differentiation. In this study, we examined the ability of primary rat marrow stromal cells at two different stages of osteoblastic differentiation to further differentiate into osteoblasts both in vitro and in vivo when seeded on titanium fiber mesh scaffolds either with or without RGD peptide tethered to the surface. In vitro, the tethered RGD peptide resulted in reduced initial cell proliferation. In vivo, there was no effect of tethered RGD peptide on ectopic bone formation in a rat subcutaneous implant model. Scaffold/cell constructs exposed to dexamethasone for 4 days prior to implantation (+dex constructs) resulted in significant bone formation whereas no bone formation was observed in--dex constructs. These results show that the osteoblastic differentiation of marrow stromal cells was not dependent on surface tethered RGD peptide, and that the initial differentiation stage of implanted cells plays an important role in bone formation in titanium fiber mesh bone tissue engineering constructs.     

Inhibition of proprotein convertases enhances cell migration and metastases development of human colon carcinoma cells in a rat model

Although proprotein convertases are involved in tumor development, nothing is known about their role in metastatic dissemination. To investigate the involvement of convertase inhibition, we used human colon carcinoma cells overexpressing alpha1-antitrypsin Portland (alpha1-PDX, PDX39P cells), a potent convertase inhibitor. We previously reported that these cells bear uncleaved integrin alpha subunits and display an altered attachment to vitronectin that is correlated with defects in the intracellular signaling pathways activated by alphavbeta5 integrin ligation. In this study, we demonstrate that the inhibition of proprotein convertase activity either by overexpression of alpha1-PDX or with the synthetic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) led to a significant increase in cell migration supported by the alphavbeta5 integrin. A collagen gel invasion assay showed that PDX39P cells also displayed an invasive ability, contrary to control cells. Moreover, when injected to immunosuppressed newborn rats, PDX39P cells were highly invasive, as they induce 10 times more metastases than mock-transfected cells. In addition, the aggressiveness of PDX39P cells can be greatly reduced by a function-blocking monoclonal antibody (mAb) against the alphav subunit. It thus seems that inhibition of proprotein convertases enhances the in vivo invasiveness of colon tumor cells likely due to an increase in cell migration mediated by alphav integrins.     

Induction of an epithelial integrin alphavbeta6 in human cytomegalovirus-infected endothelial cells leads to activation of transforming growth factor-beta1 and increased collagen production

Human cytomegalovirus (CMV) infection is a major cause of morbidity in immunosuppressed individuals, and congenital CMV infection is a leading cause of birth defects in newborns. Infection with pathogenic viral strains alters cell-cell and cell-matrix interactions, affecting extracellular matrix remodeling and endothelial cell migration. The multifunctional cytokine transforming growth factor (TGF)-beta1 regulates cell proliferation, differentiation, and extracellular matrix remodeling. Secreted as a latent protein complex, TGF-beta1 requires activation before binding to receptors that phosphorylate intracellular effectors. TGF-beta1 is activated by integrin alphavbeta6, which is strongly induced in the epithelium by injury and inflammation but has not previously been found in endothelial cells. Here, we report that CMV infection induces integrin alphavbeta6 expression in endothelial cells, leading to activation of TGF-beta1, signaling through its receptor ALK5, and phosphorylation of its intracellular effector Smad3. Infection of endothelial cells was also found to stimulate collagen synthesis through a mechanism dependent on both TGF-beta1 and integrin alphavbeta6. Immunohistochemical analysis showed integrin alphavbeta6 up-regulation in capillaries proximal to foci of CMV infection in lungs, salivary glands, uterine decidua, and injured chorionic villi of the placenta, demonstrating both its induction in endothelium and up-regulation in epithelium in vivo. Our results suggest that activation of TGF-beta1 by integrin alphavbeta6 contributes to pathological changes and may impair endothelial cell functions in tissues that are chronically infected with CMV.     

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.     

Three-dimensional synthetic niche components to control germ cell proliferation

Spermatogonial stem cells (SSCs) are increasingly studied for potential use in tissue regeneration due to their ability to dedifferentiate into embryonic stem cell-like cells. For their successful therapeutic use, these cells must first be expanded in vitro using an appropriate culture system. We hypothesized that a hydrogel with proper biochemical and biomechanical properties may mimic the composition and structure of the native basement membrane onto which SSCs reside, thus allowing us to control SSC proliferation. This hypothesis was examined in two-dimensional (2D) and three-dimensional (3D) cultures using hydrogels formed from calcium cross-linked alginate molecules conjugated with synthetic oligopeptides containing the Arg-Gly-Asp sequence (RGD peptides). The RGD peptide density (N(RGD)) in gel matrices was controlled by mixing alginate molecules modified with RGD peptides and unmodified alginate molecules at varied ratios. The mechanical stiffness was controlled with the cross-linking density of gel matrices. Interestingly, the RGD peptide density modulated cell proliferation in both 2D and 3D cultures as well as the number and size of SSC colonies formed in 3D cultures. In contrast, cell proliferation was minimally influenced by mechanical stiffness in 2D cultures. Overall, the results of this study elucidate an important factor regulating SSC proliferation and also present a bioactive hydrogel that can be used as a 3D synthetic basement membrane. In addition, the results of this study will be broadly useful in controlling the proliferation of various stem cells.     

Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports

Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.