Integrin α5ß1 promotes BMCs mobilization and differentiation to exacerbate choroidal neovascularization.

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-ß and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV.

N-Methylation of isoDGR Peptides: Discovery of a Selective α5ß1-Integrin Ligand as a Potent Tumor Imaging Agent.

Specific targeting of the integrin subtype α5ß1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective α5ß1/αvß6 peptide c(phg- isoDGR-k) into a potent peptidic RGD binding α5ß1 subtype selective ligand c(phg- isoDGR-( NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg- isoDGR-( NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring α5ß1 integrin expression in a M21 mouse xenograft.

Frontline Science: Functionally impaired geriatric CAR-T cells rescued by increased α5ß1 integrin expression.

Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5ß1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-ß1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5ß1 integrin expression. Neutralization of α5ß1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5ß1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T.

Integrin α5ß1 Inhibition by CLT-28643 Reduces Postoperative Wound Healing in a Mouse Model of Glaucoma Filtration Surgery.

Purpose: To evaluate the therapeutic potential of the small molecule integrin α5ß1 inhibitor, CLT-28643, to improve the filtering surgery outcome in a mouse model. Different dose regimens and administration routes of the inhibitor were compared with mitomycin C (MMC), the gold standard in clin ical practice. Methods: The efficacy of CLT-28643 on surgical outcome was studied in a mouse model for filtering surgery (n = 40 eyes from 20 mice per group). Single and repeated subconjunctival (SCJ) injections (1 or 2 µg) and topical eye drops (10 µg) of the integrin inhibitor were compared with 2-minute administration of MMC 0.02%. Bleb size, survival, and signs of toxicity were examined until 28 days after surgery. Immunohistochemical analysis of angiogenesis, inflammation, collagen deposition, and integrin α5ß1 expression were performed on postoperative days 3, 8, 14, and 28. A masked observer performed all the assessments. Results: Immunostaining showed that integrin α5ß1 was highly expressed in the bleb at early time-points after surgery and that CLT-28643 inhibited this upregulation. Efficacy was shown to be dose-dependent for the integrin inhibitor CLT-28643 for bleb area and survival, and the wound healing process. While 2-µg single injection of CLT-28643 improved bleb characteristics in a similar way as 10-µg administered by eye drops and MMC, repeated injections of 2 µg showed superior efficacy compared to MMC, with no corneal toxicity. Conclusions: Administration of the integrin α5ß1 inhibitor CLT-28643 has therapeutic potential as an adjunct to glaucoma surgery, possibly with a superior efficacy and tolerability compared with MMC when used at the optimal dose.

Neural crest cell-autonomous roles of fibronectin in cardiovascular development.

The chemical and mechanical properties of extracellular matrices (ECMs) modulate diverse aspects of cellular fates; however, how regional heterogeneity in ECM composition regulates developmental programs is not well understood. We discovered that fibronectin 1 (Fn1) is expressed in strikingly non-uniform patterns during mouse development, suggesting that regionalized synthesis of the ECM plays cell-specific regulatory roles during embryogenesis. To test this hypothesis, we ablated Fn1 in the neural crest (NC), a population of multi-potent progenitors expressing high levels of Fn1. We found that Fn1 synthesized by the NC mediated morphogenesis of the aortic arch artery and differentiation of NC cells into vascular smooth muscle cells (VSMCs) by regulating Notch signaling. We show that NC Fn1 signals in an NC cell-autonomous manner through integrin α5ß1 expressed by the NC, leading to activation of Notch and differentiation of VSMCs. Our data demonstrate an essential role of the localized synthesis of Fn1 in cardiovascular development and spatial regulation of Notch signaling.

Complementary, Selective PET Imaging of Integrin Subtypes α5ß1 and αvß3 Using 68Ga-Aquibeprin and 68Ga-Avebetrin.

UNLABELLED: Despite in vivo mapping of integrin αvß3 expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α5ß1 appears to be a promising target, for which purpose we designed the PET radiopharmaceutical (68)Ga-aquibeprin. METHODS: (68)Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α5ß1 integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated (68)Ga labeling. Integrin α5ß1 and αvß3 affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α5 and ß3 subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. RESULTS: (68)Ga-aquibeprin shows high selectivity for integrin α5ß1 (50% inhibition concentration [IC50] = 0.088 nM) over αvß3 (IC50 = 620 nM) and a pronounced hydrophilicity (log D = -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the ß3 integrin subunit but also an intense expression of the α5 integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. (68)Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for (68)Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer (68)Ga-avebetrin, which shows an inverse selectivity for integrin αvß3 (IC50 = 0.22 nM) over α5ß1 (IC50 = 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (∼5 mg/kg) of the respective other compound. CONCLUSION: (68)Ga-aquibeprin and (68)Ga-avebetrin are recommendable for complementary mapping of integrins α5ß1 and αvß3 by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.

CD44-mediated activation of α5ß1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and fibronectin-enriched matrices.

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of ß1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the ß1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with ß1-integrin and repressed HA-induced, CD44-mediated activation of ß1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and ß1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

Targeting effect of PEGylated liposomes modified with the Arg-Gly-Asp sequence on gastric cancer.

Previous studies have demonstrated that the α5ß1 integrin-mediated interaction with fibronectin (FN) occurs through the Arg-Gly-Asp (RGD) cell-binding sequence in repeat III10. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the US Food and Drug Administration. In the present study, we developed an RGD-modified PEGylated liposome-encapsulated ICG (RGD-PLS-ICG) system mediated by integrin. RGD was conjugated covalently to the distal end of DSPE-PEG2000-NH2 lipid by amide binding. The characteristics and stability of the prepared liposomes were assessed. In vitro, SGC7901 cells with high expression of integrin α5ß1 were selected by polymerase chain reaction (PCR) and western blotting. To confirm the targeting efficacies to gastric cancer, coumarin-6 was encapsulated as a fluorescent probe for in vitro study, and the targeting effect of RGD was detected by flow cytometry and confocal microscopy. In vivo, the bio distribution of RGD-PLS-ICG was studied by an in vivo imaging system in the tumor model. RGD-PLS-ICG and PLS-ICG had a higher UV absorbance spectrum and stability than free-ICG. Confocal microscopy and flow cytometry demonstrated that RGD-PLS-encapsulated coumarin-6 was efficiently associated with the SGC7901 cells, while limited interaction was found for the other groups. Moreover, the in vivo imaging of the liposomes indicated that RGD-PLS-ICG achieved more accumulation in the tumor tissues when compared with PLS-ICG. The significant in vitro and in vivo results suggest that RGD-PLS-ICG may be a promising fluorescent dye delivery system for targeting gastric cancer cell overexpression of integrin.

Regulation of the ITGA2 gene by epigenetic mechanisms in prostate cancer.

BACKGROUND: Integrin alpha2 beta1 (α2 ß1 ) plays an integral role in tumour cell invasion, metastasis and angiogenesis, and altered expression of the receptor has been linked to tumour prognosis in several solid tumours. However, the relationship is complex, with both increased and decreased expression associated with different stages of tumour metastases in several tumour types. The ITGA2 gene, which codes for the α2 subunit, was examined to investigate whether a large CpG island associated with its promoter region is involved in the differential expression of ITGA2 observed in prostate cancer. METHODS: Bisulphite sequencing of the ITGA2 promoter was used to assess methylation in formalin-fixed paraffin-embedded (FFPE) prostate tumour specimens and prostate cancer cell lines, PC3, 22Rv1 and LNCaP. Changes in ITGA2 mRNA expression were measured using quantitative PCR. ITGA2 functionality was interrogated using cell migration scratch assays and siRNA knockdown experiments. RESULTS: Bisulphite sequencing revealed strikingly decreased methylation at key CpG sites within the promoter of tumour samples, when compared with normal prostate tissue. Altered methylation of this CpG island is also associated with differences in expression in the non-invasive LNCaP, and the highly metastatic PC3 and 22Rv1 prostate cancer cell lines. Further bisulphite sequencing confirmed that selected CpGs were highly methylated in LNCaP cells, whilst only low levels of methylation were observed in PC3 and 22Rv1 cells, correlating with ITGA2 transcript levels. Examination of the increased expression of ITGA2 was shown to influence migratory potential via scratch assay in PC3, 22Rv1 and LNCaP cells, and was confirmed by siRNA knockdown experiments. CONCLUSIONS: Taken together, our data supports the assertion that epigenetic modification of the ITGA2 promoter is a mechanism by which ITGA2 expression is regulated.

Very late antigen-5 facilitates stromal progenitor cell differentiation into myofibroblast.

Fibrotic disease is associated with abrogated stromal cell proliferation and activity. The precise identity of the cells that drive fibrosis remains obscure, in part because of a lack of information on their lineage development. To investigate the role of an early stromal progenitor cell (SPC) on the fibrotic process, we selected for, and monitored the stages of, fibroblast development from a previously reported free-floating anchorage-independent cell (AIC) progenitor population. Our findings demonstrate that organotypic pulmonary, cardiac, and renal fibroblast commitment follows a two-step process of attachment and remodeling in culture. Cell differentiation was confirmed by the inability of SPCs to revert to the free-floating state and functional mesenchymal stem/stromal cell (MSC) differentiation into osteoblast, adipocyte, chondrocyte, and fibroblastic lineages. The myofibroblastic phenotype was reflected by actin stress-fiber formation, α-smooth muscle production, and a greater than threefold increase in proliferative activity compared with that of the progenitors. SPC-derived pulmonary myofibroblasts demonstrated a more than 300-fold increase in fibronectin-1 (Fn1), collagen, type 1, α1, integrin α-5 (Itga5), and integrin ß-1 (Itgb1) transcript levels. Very late antigen-5 (ITGA5/ITGB1) protein cluster formations were also prevalent on the differentiated cells. Normalized SPC-derived myofibroblast expression patterns reflected those of primary cultured lung myofibroblasts. Intratracheal implantation of pulmonary AICs into recipient mouse lungs resulted in donor cell FN1 production and evidence of epithelial derivation. SPC derivation into stromal tissue in vitro and in vivo and the observation that MSC and fibroblast lineages share a common ancestor could potentially lead to personalized antifibrotic therapies.

Resveratrol inhibits ovarian cancer cell adhesion to peritoneal mesothelium in vitro by modulating the production of α5ß1 integrins and hyaluronic acid.

OBJECTIVE: Resveratrol (Res) is known to inhibit adhesion of numerous malignancies though its effect on an adherence of ovarian cancer cells to peritoneal mesothelium remains undefined. METHODS: To address this issue, ovarian cancer cells (A2780, OVCAR-3, SKOV-3) were subjected to Res (10, 50, 100 µM), and then their adhesion to omentum-derived human peritoneal mesothelial cells (HPMCs) was assayed. RESULTS: The study showed that Res inhibits adhesion of all ovarian cancer cell lines investigated. More importantly, this effect was evident either when cancer cells were directly treated with Res (cell-dependent activity) or when intact cancer cells were pretreated with conditioned medium (CM) generated by their counterparts subjected to Res (medium-dependent activity). Cell-dependent activity of Res has been recognized to be linked with decreased level of cellular α5ß1 integrins which decreased functionality corresponds with reduced efficiency of cancer cell adhesion. Medium-related effects have been, in turn, associated with up-regulated secretion of soluble HA to environment (CM). The experiments with exogenous HA revealed the inverse relation between HA concentration in CM and cancer cell adhesion. When the CM from cells subjected with Res (with elevated HA) was supplemented with hyaluronidase, the restoration of cell adhesive capabilities occurred. CONCLUSIONS: Our studies evidenced that Res affects ovarian cancer cell adhesion to HPMCs by decreasing cellular α5ß1 integrin level and by increasing the secretion of HA to environment.

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5ß1 or αvß3/ß5 integrin antagonists in U87MG glioma cells.

BACKGROUND: Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5ß1 and αvß3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5ß1 and αvß3/ß5 purified integrins. METHODS: Small antagonists were either selective for α5ß1 integrin, for αvß3/ß5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5ß1 integrin in the biological assays. RESULTS: U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5ß1 or αvß3/ß5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5ß1 integrin and was inhibited selectively by α5ß1 integrin antagonists but increased by αvß3/ß5 integrin antagonists. CONCLUSIONS: We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. GENERAL SIGNIFICANCE: Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5ß1 integrin-dependent cell migration.

Increased expression of α5ß1-integrin is a prognostic marker for patients with gastric cancer.

OBJECTIVE: The study was to evaluate the association of expression level of α5ß1-integrin with clinicopathologic features and prognosis in gastric cancer (GC). METHODS: The expression of α5ß1-integrin in normal gastric mucosa and GC tissue was detected with immunohistochemistry. The level of α5 and ß1 mRNA in GC tissues and non-neoplastic tissues was evaluated in 48 paired cases by quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis by the Kaplan-Meier method was performed to assess prognostic significance. RESULTS: The α5ß1-integrin expression was detected in 68.3 % (127/186) GC samples, and there was a significant difference on their positive expression rate between GC tissue and normal gastric mucosa (P < 0.001). The positive expression rate of α5ß1-integrin in patients with poor histologic differentiation (P = 0.001), lymph node metastasis (P < 0.001), and recurrence (P < 0.001) group was heightened. Using Kaplan-Meier analysis, a comparison of survival curves of low versus high expresser of α5ß1-integrin revealed a highly significant difference in human GC tissue (P = 0.002), which suggested that overexpression of α5ß1-integrin is associated with a worse prognosis. Multivariate analyses showed that α5ß1-integrin expression was independent risk factor predicting overall survival [Hazard ratio (HR) 1.594, 95 % confidence interval (CI) 1.236-2.408, P = 0.006] and disease-free survival [HR 3.952, 95 % CI 1.676-9.861, P = 0.003] in GC. CONCLUSIONS: The α5ß1-integrin promotes angiogenesis and associates with lymph node metastasis, vascular invasion and poor prognosis of GC. The current study shows that α5ß1-integrin may be an independent prognostic factor for GC patients.

Extensive vascular remodeling in the spinal cord of pre-symptomatic experimental autoimmune encephalomyelitis mice; increased vessel expression of fibronectin and the α5ß1 integrin.

Alterations in vascular structure and function are a central component of demyelinating disease. In addition to blood-brain barrier (BBB) breakdown, which occurs early in the course of disease, recent studies have described angiogenic remodeling, both in multiple sclerosis tissue and in the mouse demyelinating model, experimental autoimmune encephalomyelitis (EAE). As the precise timing of vascular remodeling in demyelinating disease has yet to be fully defined, the purpose of the current study was to define the time-course of these events in the MOG35-55 EAE model. Quantification of endothelial cell proliferation and vessel density revealed that a large part of angiogenic remodeling in cervical spinal cord white matter occurs during the pre-symptomatic phase of EAE. At the height of vascular remodeling, blood vessels in the cervical spinal cord showed strong transient upregulation of fibronectin and the α5ß1 integrin. In vitro experiments revealed that α5 integrin inhibition reduced brain endothelial cell proliferation under inflammatory conditions. Interestingly, loss of vascular integrity was evident in all vessels during the first 4-7days post-immunization, but after 14days, was localized predominantly to venules. Taken together, our data demonstrate that extensive vascular remodeling occurs during the pre-symptomatic phase of EAE and point to a potential role for the fibronectin-α5ß1 integrin interaction in promoting vascular remodeling during demyelinating disease.

QHREDGS enhances tube formation, metabolism and survival of endothelial cells in collagen-chitosan hydrogels.

Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvß3 or α5ß1 were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction.

Bioresorbable polymersomes for targeted delivery of cisplatin.

Nontoxic bioresorbable polymersomes have been developed that efficiently and site-selectively tether targeting peptides under mild conditions with no toxic catalysts. The binding and release properties of these polymersomes have been evaluated when targeting DLD-1 human colon cancer cells overexpressing the α(5)ß(1) integrin. The delivery efficacy to these cells is markedly improved over commonly used RGD targeting peptides by use of an α(5)ß(1)-specific targeting peptide, PR_b. Release profiles in buffered solution from pH 7.4 to 4.5 were evaluated and compared to release after binding to cells, and enzymatic degradation was identified as a major cause of rapid payload release in the cell. Intracellular trafficking and release were imaged via confocal microscopy in live cells and colocalization with organelles was evaluated quantitatively over time. Finally, the anticancer drug cisplatin was encapsulated in the PR_b functionalized polymersomes and the presence of PR_b greatly improved delivery efficacy, with increased cisplatin-induced losses to targeted DLD-1 colon cancer cell viability. When delivered to CACO-2 model human epithelial cells expressing low levels of α(5)ß(1) integrin, low toxicity was maintained, suggesting that targeting was specific to α(5)ß(1) overexpressing cells. These results demonstrate that PR_b-functionalized bioresorbable polymersomes may be an attractive route to minimizing the dose-limiting side effects associated with existing approaches to cisplatin chemotherapy.

In vitro evaluation of hydroxyapatite-chitosan-gelatin composite membrane in guided tissue regeneration.

Resorbable biomaterials have been investigated as barrier membranes to compartmentalize the periodontal defects while selectively guiding osteoprogenitor cell proliferation and bone tissue expansion. Hydroxyapatite (H), chitosan (C), and gelatin (G) have chemical similarity to the structural components of natural bone and their composites have been tested as bone scaffolds. Human mesenchymal stem or stromal cells (hMSCs) are inducible osteoprogenitors and are responsible for bone tissue repair and regeneration. In this study, the dynamic interactions of hMSC with composite hydroxyapatite-chitosan-gelatin (HCG) membranes were investigated. The association of HCG formed a biodegradable membrane with ~60 wt % water and an initial stiffness of ~20 kPa. Preconditioning in serum-containing media resulted in the formation nanopores in the HCG membranes and the increase of extracellular matrix (ECM) protein adsorption. Expression of integrin α(2)ß(1) and α(5)ß(1) coincided with ECM enrichment, suggesting the enhanced cell-ECM interactions. The elevated expression of bone marker proteins and genes in the HCG membranes suggests the progression of hMSC osteogenic differentiation in the absence of chemical induction. The results showed that the HCG membranes possess sufficient mechanical and structural properties to function as a barrier membrane, and that the adsorbed ECM proteins effectively functionalized the HCG membranes and promoted hMSC osteogenic differentiation.

Influence of divalent cations on the cytoskeletal dynamics of K562 cells determined by nano-scale bead tracking.

Cytoskeletal reorganization processes can be analyzed by studying the nanometer-scale spontaneous motion of beads bound to the cytoskeleton. The bead motion is determined by force fluctuations within the cytoskeletal network that originates from myosin motor activity and dynamic restructuring of cytoskeletal filaments. We investigated to what extend the spontaneous bead motion is influenced by the dynamics of the link between the bead and the cytoskeleton in the presence of divalent cations. Our data show that, when K562 cells expressing constitutively (alpha 5 beta 1) integrin and when stably transfected with (alpha v beta 3) integrin, spontaneous bead motion is dramatically affected by the presence of 1mM Mn(2+) (integrin, activate state) compared to 1mM Ca(2+)/Mg(2+) ions (integrin, inactive state). The directionality of the bead motion, which is influenced by the overall stability of the cytoskeletal network and by actomyosin-generated forces, is markedly different, whilst the persistence remained similar due to the specific binding of either Mn(2+) or Mg(2+)/Ca(2+) ions.

Elevated levels of Lewis y and integrin α5ß1 correlate with chemotherapeutic drug resistance in epithelial ovarian carcinoma.

OBJECTIVE: To measure Lewis y and integrin α(5)ß(1) expression in epithelial ovarian carcinoma and to correlate the levels of these molecules with ovarian carcinoma chemotherapy and prognosis. METHODS: The study population included 34 ovarian carcinoma patients with chemotherapeutic drug-resistance, six partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Immunochemistry was used to determine expression of Lewis y antigen and integrin α(5)ß(1) in ovarian carcinoma tissues, and correlation of these molecules with chemotherapy resistance was further investigated, Multi-factor logistic regression analysis was applied to investigate: age, surgical stage, grade, subtype of patient cases, metastasis of lymph nodes, residual tumor size, expression levels of Lewis y antigen and integrin α(5)ß(1) correlation with ovarian carcinoma chemotherapy resistance. RESULTS: The expression rates of Lewis y antigen and integrins α(5) and ß(1) were significantly greater in the drug-resistant group (91.17%, 85.29%, 88.24%) than the partially sensitive (50.00%, 33.33%, 50.00%) or sensitive groups (61.54%, 57.69%, 55.77%). Binary logistic regression analysis revealed that surgical stage, residual tumor size, and expression of integrin α(5) and Lewis y in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance. CONCLUSIONS: Overexpression of Lewis y and integrin α(5) are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

ADAM15 to α5ß1 integrin switch in colon carcinoma cells: a late event in cancer progression associated with tumor dedifferentiation and poor prognosis.

ADAM15, a member of the A Disintegrin And Metalloproteinase (ADAM) family, is a membrane protein containing an adhesion domain that binds to α5ß1 integrin through a unique RGD domain. ADAM15, expressed by human normal colonocytes, is involved in epithelial wound healing and tissue remodeling in inflammatory bowel disease. The aims of our study were (i) to analyze ADAM15 expression in a series of colon carcinomas and paired normal mucosa and (ii) to integrate the spatial relationship of ADAM15 with its binding partners α5ß1 integrin, a mesenchymal marker, as well as with other adhesion molecules, α3ß1 integrin and E-cadherin. A series of 94 colon carcinomas of the non other specified category were graded according to the World Health Organization classification. Immunohistochemistry was performed on frozen tissue sections using antibodies directed to ADAM15, α5ß1 and α3ß1 integrins, and E-cadherin. ADAM15 was quantified at the mRNA level. Finally, promoter methylation of ADAM15 was examined as well as the microsatellite instability status (MSS/MSI). Thirty-six percent of colorectal carcinomas displayed a reduced expression of ADAM15 in cancer cells, confirmed at the mRNA level in most cases, without promoter methylation. ADAM15 down-regulation was associated with histologically poorly differentiated carcinomas. In addition, it was associated with the acquisition of α5ß1 by cancer cells and down-regulation of α3ß1 integrin and E-cadherin. Finally this profile that includes characteristic of epithelial to mesenchymal transition is a late progression event of colon cancer with a poor prognosis.