Beta1 Integrin Research Articles

Tissue Factor signaling: a multi-faceted function in biological processes

Tissue factor (TF), originally discovered to initiate coagulation, is more recently recognized to be involved in other biological processes, such as migration and anti-apoptosis. TF-mediated signaling regulates gene expression and protein synthesis, leading to alterations in cellular behavior. The proteolytic activity of factor VIIa (FVIIa), beta-1 integrin interaction and protease-activated receptor (PAR) activation are some of the key events involved in TF signaling. Post-translational modifications of TF may regulate signaling but this remains elusive. In vivo studies have established that TF signaling severely contributes to processes like angiogenesis, cancer growth and inflammation. This review focuses on the mechanism underlying TF-mediated intracellular signaling with its related physiological and mainly pathological consequences.

Acute Tumor Lysis Syndrome. Modulation of Cyclooxygenase-2, Alpha V Beta Integrin and Microsomal Prostaglandin E Synthase-1 In Human Immunodeficiency Virus-1 (HIV-1) Squamous Cell Carcinoma (SCC) of the Penis with Selenomethionine/Nsaids

AbstractAbstract 4880Invasive squamous cell carcinoma of the penis overexpress Cyclooxygenase-2 (cox-2), Microsomal Prostaglandin E Synthase-1 and Alpha V Beta Integrins. Trace minerals selenium (selenomethionine) is deficient in many neoplasia conditions especially SCC of the penis in HIV-1 patients. Selenium, an essential trace element, has been shown to inhibit tumorigenesis. Hence, selenium and arachidonic acid metabolism may be linked to SCC. Penile cancer (SCC histology) displays elevated COX-2 and PGE2 can be regulated by selenium supplementation.We present a 47 We present a 47 yo male with HIV-1, CD4-133/cumm, VL of 95,000 copies/ml with T4 advanced stage SCC of the penis post XRT, surgical skin graft with extensive lymphedema, pain and KPS score of 40–50%. CT scan of Abdomen/Pelvis was c/w extensive disease of his right inguinal region, bladder compressed and displaced into left pelvis and tissue massof the right and left groin with ill defined nodular densities within RLL and left hepatic lobe. Bone marrow was hypocellular, M:E ratio of 8:1 with foamy macrophages. When treated for selenium deficient level and pain with selenomethionine and COX-2 inhibitor, acute tumor lysis syndrome developed with hyperkalemia, hyperuricemia and hypocalcemia defervesing with aggressive therapy with hydration, alkalinization and allopurinol. Conclusion:Overexpression of COX-2, mPEGS-1, alpha V beta integrin leads to phenotypic changes in epithelial cells that could enhance tumorigenesis. Selenium supplementation and COX-2 inhibition may help regulate COX-2 alternating cellular adhesion and promote apoptosis in SCC of the penis and upon treatment of these HIV-1 patients, with heavy tumor burden, tumor lysis syndrome along with disease Invasive squamous cell carcinoma of the penis overexpress Cyclooxygenase-2 (cox-2), Microsomal Prostaglandin E Synthase-1 and Alpha V Beta Integrins. Trace minerals selenium (selenomethionine) is deficient in many neoplasia conditions and PGE2 can be regulated by selenium supplementation. especially SCC of the penis in HIV-1 patients. Selenium, an essential trace element, has been shown to inhibit tumorigenesis. Hence, selenium and arachidonic acid metabolism may be linked to SCC. Penile cancer (SCC histology) displays elevated COX-2. Disclosures:No relevant conflicts of interest to declare.

Urokinase Receptor (uPAR)-Dependent Integrin Redistribution Represents a Central Mechanism for Growth Factor Induced Endothelial Cell Migration

AbstractAbstract 2119Tumor angiogenesis is induced when the net balance of pro- and antiangiogenic molecules is tipped in favor of angiogenesis, the so called ‘angiogenic switch’. Recently, we described a mechanism how VEGF induces pro-urokinase (pro-uPA) activation, which led to uPAR-complex formation and internalization of beta-1 integrins into the endosomal compartment via LDLR-proteins such as ApoER2 or VLDLR. Thereby, uPAR plays a central role for VEGF-induced endothelial cell migration.Here, we describe that uPAR-induced integrin internalization and redistribution to the leading edge is not only limited to VEGF-induced endothelial cell migration, but plays a central role for others angiogenic growth factors such as fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF). Furthermore, we found that a hitherto undescribed binding site on domain 3 of uPAR for direct LDLR-protein interaction is required and sufficient for uPAR-dependent integrin redistribution. Interference with the uPAR/integrin internalization either by the Receptor Associated Protein (RAP) or a specific LDLR-binding site mimicking peptide (P1), the migratory response of endothelial cells towards the growth factors VEGF, HGF, FGF-2, or EGF was almost blocked (20.24% ± 4.56%). Consistently, expression of a mutated uPAR lacking interaction site for LDLR-proteins in uPAR-/- endothelial cells via a retroviral construct led to reduced invasive response towards angiogenic growth factors in vitro as well as in a Matrigel plug in vivo assay.From these data we conclude that uPAR/LDLR-protein interaction represents a central molecule in growth factor-induced endothelial cell behavior, which might open a new avenue for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Differential Expression of Alpha2 Integrin In Human Bone Marrow and Cord Blood CD34+CD38- Progenitors and Stem Cells Reconstituting NOD/SCID-IL2Rgammacnull Mice.

AbstractAbstract 2609Human primitive stem cells reside in the CD34+CD38- fraction in cord blood and bone marrow. However, there is a high level of heterogeneity in these cell fractions, and the phenotype of the rare primitive stem cells remains poorly defined. We have studied expression of integrin alpha2 chain, a member of a family of beta1 integrin cell adhesion receptors, in CD34+CD38- cells in adult bone marrow and cord blood. >90% of bone marrow CD34+CD38- cells and >95% of CD34+CD38-CD90+ cells, enriched in long-term in vivo reconstituting stem cells (Majeti et al., Cell Stem Cell 1:635, 2007) expressed the integrin alpha2 chain. In contrast, in cord blood CD34+CD38- and the CD34+CD38-CD90+ subpopulation, the integrin alpha2 chain was expressed only in 37.1+/− 5.3% and 32.2+/− 4.9% of the cells (mean+/− SD), respectively. To determine whether integrin alpha2 expression could be used to identify functionally distinct stem and progenitor cell populations in cord blood and bone marrow, we isolated CD34+CD38- integrin alpha2+ and alpha2- cells by flow cytometry and analyzed these by in vivo transplantation into immunodeficient NOD/SCID-IL2Rgammacnull (NSG) mice and by in vitro progenitor cell assays (long-term culture initiating cell, LTC-IC, and colony assays). Transplantation of cord blood CD34+CD38- integrin alpha2+ cells resulted in significantly higher level of human CD45+ (p<0.05), myeloid (p<0.01) and CD34+ (p<0.05) cell engraftment at 16–18 weeks after transplantation than integrin alpha2- cells (reconstitution/300 cells in age and sex matched recipients, Figure 1). In contrast, there were no differences in reconstitution at 12 weeks in mice transplanted with cord blood CD34+CD38- alpha2+ and alpha2- cells. Because of lower engraftment capacity of adult bone marrow cells in immunodeficient mice, bone marrow CD34+CD38- cells were analyzed after intra-bone transplantation. After 12 weeks only few mice transplanted with the CD34+CD38- integrin alpha2+ cells but none transplanted with the corresponding alpha2- cells were reconstituted with human CD45+ or myeloid cells at a level '0.1%. The LTC-IC progenitors within the CD34+CD38- populations, assayed after 6 weeks culture on stroma, in adult bone marrow were highly enriched in the alpha2+ as compared with alpha2- fraction (mean +/− SD 96.7+/− 57.0 and 0.2+/− 0.6 colonies/50 cells, respectively), whereas in cord blood they resided equally in both integrin alpha2+ and alpha2- cell fractions (mean +/− SD 77.4+/− 59.4 and 108.8+/− 96.0 colonies/50 cells, respectively). The lineage committed CFU-GM and BFU-E progenitors in adult bone marrow were within integrin CD34+CD38- alpha2+ and alpha2- fractions (mean +/− SD 8.5+/− 5.3 and 14.9+/− 16.2 colonies/100 cells, respectively), whereas in cord blood they were significantly enriched in the alpha2- fraction (mean +/− SD 9.3+/− 5.5 and 22.0+/− 6.7 colonies/100 cells in alpha2+ and alpha2- fractions, respectively). Taken together, our results show expression of integrin alpha2 receptor in most of the primitive cord blood long-term in vivo reconstituting stem cells, with a gradual loss of the integrin alpha2 receptor during maturation to short term in vivo reconstituting stem cells, LTC-IC and lineage committed progenitors. Furthermore, our findings show distinct ontogeny-related differences in the expression of the integrin alpha2 receptor in the functionally defined primitive and lineage-committed CD34+CD38- progenitors, indicating differences in cellular interactions of cord blood and bone marrow progenitors with the hematopoietic niches. [Display omitted] Disclosures:Ekblom:Bristol-Myers Squibb: Honoraria.

Long-Term Effects of In Vivo Administration of An Anti-VLA-4 Antibody (natalizumab) on Mobilization of Committed and Primitive Hematopoietic Progenitor Cells In Humans

AbstractAbstract 2264The alpha4 beta1 integrin (VLA-4) exert a critical role on hematopoiesis by confining hematopoietic stem cells (HSC) and progenitor cells (HPC) within the niche. Previous preclinical studies have pointed out this role showing that HPCs can be mobilized by in vivo administration of a blocking anti-VLA-4 antibody (Ab) (Papayannopoulou et al, 1993). Very recently, two papers (Bonig et al, 2008; Zohren et al, 2008) have shown that such Ab exhibits a similar effect in humans for one month after treatment by natalizumab. In the present study, we have investigated long-term hematopoietic effects (up to 23 months) of repeated infusions of natalizumab in patients treated for multiple sclerosis (n=22). Seven patients have been explored sequentially (every month for one year) and 15 have been studied punctually (6 before and 9 after one year of treatment). We found that peripheral blood leukocytosis was consistently increased for one year in relation to lymphocytes, particularly to B lineage (by 3-fold). In parallel, an increase of circulating HPCs (CD34+ cells and total CFU) was observed and appeared more pronounced with levels above baseline values in all the patients studied (by 6-fold) while no increase of circulating mesenchymal stromal cells (CFU-F) was found. The increase was noted for both lymphoid (T and B lineages) and myeloid (granulo-monocyte, erythroid, and megakaryocyte) committed progenitor cells but also for primitive HPC (CD34+CD38- cells and CAFC). This effect was still found at long-term (up to two years of treatment) for both committed and primitive HPC. In conclusion, the HPC mobilizing effect of chronic administration of anti-VLA-4 Ab in humans involves all types of HPCs (lymphoid and myeloid, committed and primitive ones) and is not exhausted with time. Disclosures:No relevant conflicts of interest to declare.

Vascular Recruitment of Human Retinoblastoma Cells by Multi-Cellular Adhesive Interactions with Circulating Leukocytes.

Retinoblastoma (RB) is a retinal cancer of childhood. RB survivors tend to develop additional tumors later in life, although the physical mechanisms of RB metastatic spread are largely unknown. One step in metastasis through the blood stream is tumor cell adherence to the blood vessel wall through specific receptor:ligand interactions. Yet, human RB cell lines RB143 and WERI-Rb27 do not express selectin ligands or beta-2 integrins and cannot directly interact with inflamed endothelium. In this study, we show that RB cells express ICAM-1, a beta-2 integrin ligand that correlates with metastasis and is preferentially co-expressed on RB cells that also express ABCG2, a stem cell marker associated with chemoresistance and metastasis. Based on the presence of ICAM-1+ RB cells, we tested the hypothesis that RB cells could be recruited to an E-selectin surface via attachment to activated polymorphonuclear cells (PMNs). We characterized the dynamic adhesion between RB cells and PMNs within E-selectin coated microtubes under a physiological range of wall shear stress values (0.2-5 dyn/cm2). We show that activated PMNs are necessary for the recruitment of RB cells through ICAM-1:LFA-1 binding. Results from this work may lead to new strategies that target the metastatic spread of tumor cells.

Laminin-511 and integrin beta-1 in hair follicle development and basal cell carcinoma formation

BackgroundInitiation of the hair follicle placode and its subsequent growth, maturation and cycling in post-natal skin requires signaling interactions between epithelial cells and adjacent dermal cells and involves Shh signaling via the primary cilium. Previous reports have implicated laminins in hair follicle epithelial invagination.ResultsHere we use a human BCC model system and mouse mutants to re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development.ConclusionsOur data provides support for a primary role of laminin-511 promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.

CD103 in the development of experimental asthma

Objective/purpose CD103 (Alpha-E Beta-7 integrin) is expressed on various cell types involved in the development of asthma, including dendritic cells (DCs) and CD4 and CD8 T regulatory cells. This integrin binds E-cadherin on epithelial cells and plays a role in regulating the migration and proliferation of cells. Also, CD103 dendritic cells have been reported to direct the development of naive T cells into T regulatory cells. However, little is known about the exact role of CD103 in the development of asthma. The objective of this project is to investigate the role of this integrin in asthma pathogenesis, in the hope of finding new molecular targets for the treatment of asthma.

The cross-talk between the urokinase receptor and fMLP receptors regulates the activity of the CXCR4 chemokine receptor.

The receptor (CXCR4) for the stromal-derived factor-1 (SDF1) and the urokinase-receptor (uPAR) are up-regulated in various tumors. We show that CXCR4-transfected cells migrate toward SDF1 on collagen (CG) and do not on vitronectin (VN). Co-expression of cell-surface uPAR, which is a VN receptor, impairs SDF1-induced migration on CG and allows migration on VN. Blocking fMLP receptors (fMLP-R), alpha-v integrins or the uPAR region capable to interact with fMLP-Rs, impairs migration of uPAR/CXCR4-transfected cells on VN and restores their migration on CG. uPAR co-expression also reduces the adherence of CXCR4-expressing cells to various components of the extracellular matrix (ECM) and influences the partitioning of beta1 and alpha-v integrins to membrane lipid-rafts, affecting ECM-dependent signaling. uPAR interference in CXCR4 activity has been confirmed in cells from prostate carcinoma. Our results demonstrate that uPAR expression regulates the adhesive and migratory ability of CXCR4-expressing cells through a mechanism involving fMLP receptors and alpha-v integrins.

The PhScN Peptide as a Potent Agent for Targeting Both Invasion and Survival and for Increasing Radiosensitivity in Human Prostate Cancer

Purpose/Objectives(s): Metastatic invasion is caused by constitutive activation of the alpha5 beta1 integrin fibronectin receptor (FnR), and is blocked by the PHSCN peptide (Ac-PHSCN-NH2) in both preclinical models and in Phase 1 clinical trial. An endoproteinase-resistant derivative of Ac-PHSCN-NH2 was made by including D-isomers of Histidine and Cysteine to form the PhScN peptide: Ac-PhScN-NH2. Ac-PhScN-NH2 is a highly potent inhibitor of alpha5 beta1 integrin-mediated invasion and survival of human prostate cancer cells, both in vitro and in vivo.

Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development

The integrin-linked kinase (ILK), pinch and parvin ternary complex connects the cytoplasmic tails of beta1 integrins to the actin cytoskeleton. We recently showed that constitutive expression of ILK and alpha parvin in both the ureteric bud and the metanephric mesenchyme of the kidney is required for kidney development. In this study, we define the selective role of ILK in the ureteric bud of the mouse kidney in renal development by deleting it in the ureteric cell lineage before the onset of branching morphogenesis (E10.5). Although deleting ILK resulted in only a moderate decrease in branching, the mice died at 8 weeks of age from obstruction due to the unprecedented finding of intraluminal collecting duct cellular proliferation. ILK deletion in the ureteric bud resulted in the inability of collecting duct cells to undergo contact inhibition and to activate p38 mitogen-activated protein kinase (MAPK) in vivo and in vitro. p38 MAPK activation was not dependent on the kinase activity of ILK. Thus, we conclude that ILK plays a crucial role in activating p38 MAPK, which regulates cell cycle arrest of epithelial cells in renal tubulogenesis.

The chemokine receptor 7 regulates cell adhesion and migration via β1 integrin in metastatic squamous cell carcinoma of the head and neck

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Chemokine receptor 7 (CCR7) has been shown to regulate integrin which can then facilitate adhesion of cancer cells to and/or migration through the extracellular matrix (ECM). In order to identify the connection between CCR7 and beta1 integrin, and the influence on cell adhesion and migration in metastatic squamous cell carcinoma of the head and neck (SCCHN). We use adhesion assays, migration assay, immunofluorescence staining, western blotting, and immunohistochemical analysis to find whether beta1 integrin can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and beta1 integrin inhibitors RGD-peptide. Our results demonstrate that CCR7 favours PCI-37B cell adhesion and migration, and induces reorganization of the actin cytoskeleton and up-expression of beta1 integrin protein. beta1 integrin inhibitor RGD-peptide can block all these effects. Taken together, our data indicate that CCR7 regulate cell adhesion and migration via beta1 integrin in metastatic SCCHN, and these results can provide a basis for new strategies in preventing metastases of SCCHN.

TPA Is a Potent Mitogen for Renal Interstitial Fibroblasts: Role of β1 Integrin/Focal Adhesion Kinase Signaling

Proliferation and expansion of interstitial fibroblasts are predominant features of progressive chronic kidney diseases. However, how interstitial fibroblast proliferation is controlled remains ambiguous. Here we show that tissue-type plasminogen activator (tPA) is a potent mitogen that promotes interstitial fibroblast proliferation through a cascade of signaling events. In vitro, tPA promoted cell proliferation of rat kidney fibroblasts (NRK-49F), as assessed by cell counting, cell proliferation assay, and bromodeoxyuridine labeling. tPA also accelerated NRK-49F cell cycle progression. Fibroblast proliferation induced by tPA was associated with an increased expression of numerous proliferation-related genes, including c-fos, c-myc, proliferating cell nuclear antigen, and cyclin D1. The mitogenic effect of tPA was independent of its protease activity, but required LDL receptor-related protein 1. Interestingly, inhibition of beta1 integrin signaling prevented tPA-mediated fibroblast proliferation. tPA rapidly induced tyrosine phosphorylation of focal adhesion kinase (FAK), which led to activation of its downstream mitogen-activated protein kinase signaling. Blockade of FAK, but not integrin-linked kinase, abolished the tPA-triggered extracellular signal-regulated protein kinase 1/2 activation, proliferation-related gene induction, and fibroblast proliferation. In vivo, proliferation of interstitial myofibroblasts in tPA null mice was attenuated after obstructive injury, compared with the wild-type controls. These studies illustrate that tPA is a potent mitogen that promotes renal interstitial fibroblast proliferation through LDL receptor-related protein 1-mediated beta1 integrin and FAK signaling.

BRCA1 Basal-like Breast Cancers Originate from Luminal Epithelial Progenitors and Not from Basal Stem Cells

Breast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies.

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of beta1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL-induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited RANKL-induced cells migration and integrin up-regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activation of beta1 integrin and contributing to the migration of human chondrosarcoma cells.

The formin FRL1 (FMNL1) is an essential component of macrophage podosomes

Podosomes are highly dynamic actin-rich adhesion structures in cells of myeloid lineage and some transformed cells. Unlike transformed mesenchymal cell types, podosomes are the sole adhesion structure in macrophage and thus mediate all contact with adhesion substrate, including movement through complex tissues for immune surveillance. The existence of podosomes in inflammatory macrophages and transformed cell types suggest an important role in tissue invasion. The proteome, assembly, and maintenance of podosomes are emerging, but remain incompletely defined. Previously, we reported a formin homology sequence and actin assembly activity in association with macrophage beta-3 integrin. In this study we demonstrate by quantitative reverse transcriptase polymerase chain reaction and Western blotting that the formin FRL1 is specifically upregulated during monocyte differentiation to macrophages. We show that the formin FRL1 localizes to the actin-rich cores of primary macrophage podosomes. FRL1 co-precipitates with beta-3 integrin and both fixed and live cell fluorescence microscopy show that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes. Targeted disruption of FRL1 by siRNA results in reduced cell adhesion and disruption of podosome dynamics. Our data suggest that FRL1 is responsible for modifying actin at the macrophage podosome and may be involved in actin cytoskeleton dynamics during adhesion and migration within tissues.

Binding of the fibronectin-mimetic peptide, PR_b, to alpha5beta1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes.

Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses imposed during islet isolation and transplantation decrease islet viability, leading to loss of graft function. In this study, we investigated the ability of a fibronectin-mimetic peptide, PR_b, which specifically binds to the alpha(5)beta(1) integrin, to re-establish lost extracellular matrix (ECM) around isolated pig islets and increase internalization of liposomes. Confocal microscopy and Western blotting were used to show the presence of the integrin alpha(5)beta(1) on the pig islets on day 0 (day of isolation) as well as on different days of islet culture. Islets cultured in medium supplemented with free PR_b for 48 h were found to have increased levels of ECM fibronectin secretion compared to islets in normal culture conditions. Using confocal microscopy and flow cytometry, we found that PR_b peptide-amphiphile functionalized liposomes delivered to the pig islets internalized into the cells in a PR_b concentration dependent manner and nonfunctionalized liposomes showed minimal internalization. These studies proved that the fibronectin-mimetic peptide, PR_b, is an appropriate peptide bullet for applications involving alpha(5)beta(1) expressing pig islet cells. Fibronectin production stimulated through alpha(5)beta(1) PR_b binding may decrease apoptosis and therefore increase islet viability in culture. In addition, PR_b peptide-amphiphile functionalized liposomes may be used for targeted delivery of different agents to pig islet cells.

Epidermal and dermal integration into sphere‐templated porous poly(2‐hydroxyethyl methacrylate) implants in mice

Percutaneous medical devices remain susceptible to infection and failure. We hypothesize that healing of the skin into the percutaneous device will provide a seal, preventing bacterial attachment, biofilm formation, and subsequent device failure. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] with sphere-templated pores (40 microm) and interconnecting throats (16 microm) were implanted in normal C57BL/6 mice for 7, 14, and 28 days. Poly(HEMA) was either untreated, keeping the surface nonadhesive for cells and proteins, or modified with carbonyldiimidazole (CDI) or CDI reacted with laminin 332 to enhance adhesion. No clinical signs of infection were observed. Epidermal and dermal response within the poly(HEMA) pores was evaluated using light and transmission electron microscopy. Cells (keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and basement membrane proteins (laminin 332, beta4 integrin, type VII collagen) could be demonstrated within the poly(HEMA) pores of all implants. Blood vessels and dermal collagen bundles were evident in all of the 14- and 28-day implants. Fibrous capsule formation and permigration were not observed. Sphere-templated polymers with 40 microm pores demonstrate an ability to recapitulate key elements of both the dermal and the epidermal layers of skin. Our morphological findings indicate that the implant model can be used to study the effects of biomaterial pore size, pore interconnect (throat) size, and surface treatments on cutaneous biointegration. Further, this model may be used for bacterial challenge studies.

Roles of Integrins in Human Induced Pluripotent Stem Cell Growth on Matrigel and Vitronectin

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Antagonistic Anti-urokinase Plasminogen Activator Receptor (uPAR) Antibodies Significantly Inhibit uPAR-mediated Cellular Signaling and Migration

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.