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PubMed2004

Cellular growth inhibition by TGF-beta1 involves IRS proteins.

In Mv1Lu cells, insulin partially reverses transforming growth factor-beta1 (TGF-beta1) growth inhibition in the presence of alpha5beta1 integrin antagonists. TGF-beta1 appears to induce phosphorylation of IRS-2 in these cells; this is inhibited by a TGF-beta antagonist known to reverse TGF-beta growth inhibition. Stable transfection of 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by TGF-beta1) with IRS-1 or IRS-2 cDNA confers sensitivity to growth inhibition by TGF-beta1; this IRS-mediated growth inhibition can be partially reversed by insulin in 32D cells stably expressing IRS-2 and the insulin receptor (IR). These results suggest that growth inhibition by TGF-beta1 involves IRS proteins.

Animals↗
PubMed2004

Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells.

BACKGROUND: Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium. METHODS: Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured. RESULTS: As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration. CONCLUSIONS: VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Adult↗
PubMed2006

Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells.

A central question in muscle biology is how costameres are formed and become aligned with underlying myofibrils in mature tissues. Costameres are composed of focal adhesion proteins, including vinculin and paxillin, and anchor myofibril Z-bands to the sarcolemma. In the present study, we investigated the process of costamere formation ("costamerogenesis") in differentiating primary mouse myoblasts. Using vinculin and paxillin as costameric markers, we found that two additional focal adhesion components, alpha5beta1 integrin and focal adhesion kinase (FAK), are associated with costameres. We have characterized costamerogenesis as occurring in three distinct stages based on the organizational pattern of these costameric proteins. We show that both costamerogenesis and myofibrillogenesis are initiated at sites of membrane contacts with the extracellular matrix and that their maturation is tightly coupled. To test the importance of FAK signaling in these processes, we analyzed cells expressing a dominant negative form of FAK (dnFAK). When cells expressing dnFAK were induced to differentiate, both costamerogenesis and myofibrillogenesis were disrupted although the expression of constituent proteins was not inhibited. Likewise, inhibiting FAK activity by reducing FAK levels using an siRNA approach also resulted in an inhibition of costamerogenesis and myofibrillogenesis. The relationship between costamere and myofibril formation was tested further by treating myotube cultures with potassium or tetrodotoxin to block contraction and disrupt myofibril organization. This also resulted in inhibition of costamere maturation. We present a model of costamerogenesis whereby signaling through FAK is essential for both normal costamerogenesis and normal myofibrillogenesis which are tightly coupled during skeletal myogenesis.

Animals↗
PubMed2002

Validation and quantitation of an in vitro M-cell model.

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyer's patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyer's patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyer's patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.

Alkaline Phosphatase↗
PubMed2003

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition.

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.

Amino Acid Sequence↗
PubMed2004

Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels.

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.

Cell Adhesion↗
PubMed2002

A colorimetric bead-binding assay for detection of intermolecular interactions.

We have developed a new technique that rapidly and reproducibly allows direct visualization of molecular interactions, including receptor-ligand binding. The technique can be easily applied to examine binding between proteins and glycoproteins, or proteins and glycolipids, including gangliosides. In this novel bead-binding assay, the suspected 'ligand' molecule is bound to 0.2- or 1.0- micro m colored FluoSphere beads. These coated beads are then mixed directly with 150- micro m Sepharose or 50- to 75- micro m agarose beads coated with the second 'target' molecule. Binding between molecules is easily detected by immunofluoresence microscopy as colored rosettes or aggregations formed by clustering of the smaller fluorescent beads around the larger non-fluorescent bead. The validity of this technique for glycolipid binding to protein was verified through demonstration of the known interaction between the beta subunit of cholera toxin with ganglioside GM1. The bead-binding technique facilitated the novel observations of interaction between ganglioside GT1b with the alpha5 subunit of alpha5beta1 integrin and the interaction of GM3 with the epidermal growth factor receptor. A modification of this technique, in which the coated beads are bound to protein fixed on plates, allows a quantifiable colorimetric assay of interaction. This versatile and rapid technique will have widespread applications for in vitro systems and may also be useful for in vivo analysis of binding to cell surface receptor molecules.

Animals↗
PubMed2003

TGF-beta1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-beta1-enhanced cell migration in SMMC-7721 cells.

Transforming growth factor-b1 (TGF-beta1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-beta1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-beta1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-beta1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-beta1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-beta1 enhanced the expression of alpha5beta1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-beta1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-beta1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-beta1 treatment. Furthermore, TGF-beta1 induced the down-regulation of E-cadherin and the nuclear translocation of beta-catenin. These results indicated that TGF-beta1-promoted cell adhesion and TGF-beta1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-beta1-enhanced cell migration.

Carcinoma, Hepatocellular↗
PubMed2006

Low-energy helium-neon laser induces locomotion of the immature melanoblasts and promotes melanogenesis of the more differentiated melanoblasts: recapitulation of vitiligo repigmentation in vitro.

Helium-neon laser (He-Ne Laser, 632.8 nm) is a low-energy laser that has therapeutic efficacy on various clinical conditions. Our previous study has demonstrated efficacy of He-Ne laser on vitiligo, a disease characterized by skin depigmentation. To regain skin tone on vitiligo lesions, the process began by the migration of the immature melanoblasts (MBs) to the epidermis, which was followed by their functional development to produce melanin. In this study, we investigated the physiologic effects of He-Ne laser irradiation on two MB cell lines: the immature NCCmelb4 and the more differentiated NCCmelan5. The intricate interactions between MBs with their innate extracelluar matrix, fibronectin, were also addressed. Our results showed that He-Ne laser irradiation enhanced NCCmelb4 mobility via enhanced phosphorylated focal adhesion kinase expression and promoted melanogenesis in NCCmelan5. In addition, He-Ne laser decreased the affinity between NCCmelb4 and fibronectin, whereas the attachment of NCCmelan5 to fibronectin increased. The alpha5beta1 integrin expression on NCCmelb4 cells was enhanced by He-Ne laser. In conclusion, we have demonstrated that He-Ne laser induced different physiologic changes on MBs at different maturation stages and recapitulated the early events during vitiligo repigmentation process brought upon by He-Ne laser in vitro.

Animals↗
PubMed1997

A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase.

Enzymatic and immunochemical assays show a phosphatidylinositol 4-kinase in novel and specific complexes with proteins (CD63 and CD81) of the transmembrane 4 superfamily (TM4SF) and an integrin (alpha3beta1). The size (55 kDa) and other properties of the phosphatidylinositol 4-kinase (PI 4-K) (stimulated by nonionic detergent, inhibited by adenosine, inhibited by monoclonal antibody 4CG5) are consistent with PI 4-K type II. Not only was PI 4-K associated with alpha3beta1-CD63 complexes in alpha3-transfected K562 cells, but also it could be co-purified from CD63 in untransfected K562 cells lacking alpha3beta1. Thus, TM4SF proteins may link PI 4-K activity to the alpha3beta1 integrin. The alpha5beta1 integrin, which does not associate with TM4SF proteins, was not associated with PI 4-K. Notably, alpha3beta1-CD63-CD81-PI 4-K complexes are located in focal complexes at the cell periphery rather than in focal adhesions. The novel linkage between integrins, transmembrane 4 proteins, and phosphoinositide signaling at the cell periphery may play a key role in cell motility and provides a signaling pathway distinct from conventional integrin signaling through focal adhesion kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed2006

Fibronectin matrix assembly requires distinct contributions from Rho kinases I and -II.

Extracellular matrix is integral to tissue architecture and regulates many aspects of cell behavior. Fibronectin matrix assembly involves the actin cytoskeleton and the small GTPase RhoA, but downstream signaling is not understood. Here, down-regulation of either rho kinase isoform (ROCK I or -II) by small interfering RNA treatment blocked fibronectin matrix assembly, although the phenotypes were distinct and despite persistence of the alternate kinase. Remnant fibronectin on ROCK-deficient fibroblasts was mostly punctate and more deoxycholate soluble compared with controls. Fibronectin matrix assembly defects in ROCK-deficient cells did not result from decreased synthesis/secretion, altered fibronectin mRNA splicing, metalloproteinase activity, or alpha5beta1 integrin dysfunction. Rescue could be effected by ROCK protein restoration or phosphomimetic myosin light chain expression. However, the effect of ROCK I deficiency on fibronectin matrix assembly was secondary to altered cell surface morphology, rich in filopodia, resulting from high GTP-Cdc42 levels. Total internal reflection microscopy revealed that a submembranous pool of myosin light chain in control cells was missing in ROCK II-deficient cells and replaced by stress fibers. Together, two rho kinases contribute to fibronectin matrix assembly in a different manner and cortical myosin II-driven contractility, but not stress fibers, may be critical in this activity.

Animals↗
PubMed2004

Expression of integrins in intraductal papillary-mucinous tumors of the pancreas as an indicator of malignancy.

INTRODUCTION AND AIMS: Intraductal papillary-mucinous tumors (IPMTs) of the pancreas are intraductal tumors with diffuse or segmental dilation of the pancreatic ducts and intraductal papillary growth with abundant mucous secretion. We examined clinicopathological features and immunohistochemical findings to investigate the malignancy of IPMTs. METHODOLOGY: Between April 1994 and December 2002, 23 patients with IPMT underwent pancreatic resections at Nagoya City University Hospital, Japan. We immunohistochemically analyzed the expression of p53 protein, proliferating cell nuclear antigen, alpha6-integrin subunit, alpha5beta1 integrin, and interleukin-1 receptor type I in tumor specimens from the 23 patients with IPMT. RESULTS: The tumors were classified as intraductal papillary adenoma (n = 16), intraductal papillary adenocarcinoma and moderate dysplasia (n = 7). At a median follow-up of 42.9 months, 2 patients had died of this disease. The actuarial 5-year disease-free survival rate was 80.7%. Expression of the alpha6-integrin subunit was significantly strong in adenocarcinoma and moderate dysplasia tissues of IPMTs (P = 0.038). CONCLUSIONS: Our current results indicate that alpha6-containing integrin expression can be a significant marker of malignancy in IPMTs. We emphasize that immunohistochemical investigation of resected specimens is indispensable in cases of IPMT, so that appropriate postoperative treatments for malignant IPMTs are initiated to improve prognosis.

Adenocarcinoma, Mucinous↗
PubMed2003

Active detachment involves inhibition of cell-matrix contacts of malignant melanoma cells by secretion of melanoma inhibitory activity.

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells. Recent results revealed a direct interaction of MIA and epitopes within extracellular matrix proteins including fibronectin. The aim of this study was to analyze functional consequences mediated by this interaction. Here we show that MIA interferes specifically with attachment of melanoma cells to fibronectin, a phenomenon we refer to as active detachment. Antibodies inhibiting binding of alpha4beta1 and alpha5beta1 integrins to fibronectin cross-react specifically with MIA, suggesting that MIA shares significant structural homology with the binding pockets of these integrins and thereby masks the respective epitopes on extracellular matrix molecules. Several peptides derived from fibronectin and from a phage display screening were tested with respect to a potential MIA-inhibitory effect. In vitro tests identified two peptides affecting MIA function; both inhibited growth of melanoma metastases in vivo. In summary, we conclude that MIA may play a role in tumor progression and spread of malignant melanomas via mediating active detachment of cells from extracellular matrix molecules within their local milieu. Further, our results suggest that inhibiting MIA functions in vivo may provide a novel therapeutic strategy for metastatic melanoma disease.

Amino Acid Sequence↗
PubMed2006

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype.

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.

Abscess↗
PubMed2004

Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion.

We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the alpha5beta1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positive bacteria. We also observed that extracting membrane cholesterol with methyl-beta-cyclodextrin (MBCD) did not affect the recruitment of CD55, GM1, or beta1 integrin to adhering Dr-positive bacteria. In contrast, extracting or changing membrane-bound cholesterol by means of drugs that modify lipid rafts (MBCD, filipin III, or mevalonate plus lovastatin plus MBCD) inhibited the entry of Dr-positive IH11128 both into cells that expressed VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) and into those that did not (parental Caco-2 cells). Finally, restoring cholesterol within the cell membrane of MBCD-treated cells restored Dr-positive IH11128 internalization.

Actins↗
PubMed2003

Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML development.

We have previously found that P210BCR-ABL increases the adhesion of hematopoietic cell lines to fibronectin by a mechanism that is independent of tyrosine kinase activity. To investigate the pathway(s) by which P210BCR-ABL influences cell adhesion, we used a quantitative cell adhesion device that can discern small changes in cell adhesion to assay P210BCR-ABL with mutations in several critical domains. We expressed P210BCR-ABL mutants in 32D myeloblast cells and found that binding to fibronectin is mediated primarily by the alpha5beta1 integrin. We performed a structure/function analysis to map domains important for cell adhesion. Increased adhesion was mediated by 3 domains: (1) the N-terminal coiled-coil domain that facilitates oligomerization and F-actin localization; (2) bcr sequences between aa 163 to 210; and (3) F-actin localization through the C-terminal actin-binding domain of c-abl. We compared our adhesion results with the ability of these mutants to cause a chronic myelogenous leukemia (CML)-like disease in a murine bone marrow transplantation assay and found that adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion but that it is dispensable for the development of CML.

Actins↗
PubMed2005

Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti-MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)-stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-alpha (TNF-alpha)-activated endothelium is also inhibited by anti-MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1-stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on alpha4beta1 and alpha5beta1 integrins. Binding of monocytes to TNF-alpha-activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation.

Cell Movement↗
PubMed2004

Expression and function of homing-essential molecules and enhanced in vivo homing ability of human peripheral blood-derived hematopoietic progenitor cells after stimulation with stem cell factor.

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the alpha4beta1 and alpha5beta1 integrins very-late antigen (VLA)-4 and VLA-5 on peripheral blood-derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA-4 and VLA-5 on CD34+/c-kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient-derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long-term culture-initiating cell. The improvement of the homing abilities of SCF-stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic-scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.

Antigens, CD↗