Opticin Ameliorates Hypoxia-Induced Retinal Angiogenesis by Suppression of Integrin α2-I Domain-Collagen Complex Formation and RhoA/ROCK1 Signaling.

Purpose: It was previously demonstrated that opticin (OPTC) inhibits the collagen-induced promotion of bioactivities of human retinal vascular endothelial cells (hRVECs). The present in vivo study aimed to further investigate the regulatory role of opticin in vitreous collagen-mediated retinal neovascularization and to elucidate its regulatory mechanisms with regard to integrin α2-I domain-GXXGER complex formation and RhoA/ROCK1 signal change. The regulatory role of Mg2+ on integrin α2-I domain-GXXGER complex formation in the above process was also investigated. Methods: The zebrafish model of hypoxia-induced retinopathy was established, and OPTC-overexpressing plasmids were intravitreally injected to assess the antiangiogenesis effect of opticin. The regulatory role of opticin in integrin α2-I domain-GXXGER complex formation in vivo was analyzed by mass spectrometry. The mRNA and protein expression of RhoA/ROCK1 were examined. The concentration of Mg2+ as an activator of the integrin α2-I domain-GXXGER complex was measured. Solid-phase binding assays were performed to investigate the interference of opticin in integrin α2 collagen binding and the regulatory role of Mg2+ in that process. Results: Opticin and OPTC-overexpressing plasmid injection reduced retinal neovascularization in the zebrafish model of hypoxia-induced retinopathy. Mass spectrometry revealed that opticin could inhibit integrin α2-I domain-GXXGER complex formation. The Mg2+ concentration was also decreased by opticin, which was another indication of the complex activation. Injection of OPTC-overexpressing plasmids inhibited mRNA and the protein expression of RhoA/ROCK1 in the zebrafish model of hypoxia-induced retinopathy. The solid-phase binding assay revealed that opticin could block integrin α2-collagen I binding in the presence of Mg2+. Conclusions: Opticin exerts its antiangiogenesis effect by interfering in the Mg2+-modulated integrin α2-I domain-collagen complex formation and suppressing the downstream RhoA/ ROCK1 signaling pathway.

Integrin α2ß1 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gel.

Although atelocollagen gel is used as a scaffold for culturing human articular cartilage-derived chondrocytes, little is known about cell-gel interactions. In this study, we investigated the mechanism via which atelocollagen gel affects human articular cartilage-derived chondrocytes. Two types of three-dimensional cultures of human articular cartilage-derived chondrocytes (i.e., with and without atelocollagen gel) were compared. While the amount of atelocollagen gel in culture gradually decreased with time, it promoted the expression of matrix metalloproteinases (MMPs) during the early stages of culture. Genome-wide differential gene expression analysis revealed that cell membrane- and extracellular matrix-related genes were highly ranked among up- and down-regulated groups in cells cultured in the presence of atelocollagen gel. Among the integrin family of genes, the expression of integrin subunit alpha 2 and integrin subunit alpha 10 was significantly increased in the presence of atelocollagen gel. Blocking α2ß1 integrin with the specific inhibitor BTT 3033 had a significant effect on cell proliferation, MMP expression, and cell shape, as well as on the response to mechanical stimulation. Taken together, our findings indicate that the α2ß1 integrin pathway plays an important role in the interaction of atelocollagen gel with human articular cartilage-derived chondrocytes and may be a potential therapeutic target for articular cartilage disorders.

microRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation, migration, and invasion, while promoting apoptosis of colorectal cancer cells by downregulating ITGA2.

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2.

Interleukin-4 induces a CD44high /CD49bhigh PC3 subpopulation with tumor-initiating characteristics.

Pro- and anti-inflammatory cytokines may influence proliferation, migration, invasion, and other cellular events of prostate cancer (PCa) cells. The hyaluronan receptor CD44, which is regulated by Interleukin (IL)-4, is a prostate basal cell marker. CD44high /CD49bhigh expressing cells have been demonstrated to have tumor-initiating characteristics. Here, we aimed to analyze the effects of long-term IL-4 treatment on CD44/CD49b expression, migration, proliferation, and clonogenic potential of basal-like PCa cells. To this end PC3 cells were treated over 30 passages with 5 ng/mL IL-4 (PC3-IL4) resulting in an increased population of CD44high expressing cells. This was concurrent with a clonal outgrowth of cuboid-shaped cells, with increased size and light absorbance properties. Flow cytometry revealed that the PC3-IL4 CD44high expressing subpopulation corresponds to the CD49bhigh population. Isolation of the PC3-IL4 CD44high /CD49bhigh subpopulation via fluorescence-associated cell sorting showed increased migrative, proliferative, and clonogenic potential compared to the CD44low /CD49blow subpopulation. In conclusion, IL-4 increases a PC3 subpopulation with tumor-initiating characteristics. Thus, IL-4, similar to other cytokines may be a regulator of tumor-initiation and hence, may present a suitable therapy target in combination with current treatment options.

Identification of curcumin-inhibited extracellular matrix receptors in non-small cell lung cancer A549 cells by RNA sequencing.

Curcumin is a potent anti-cancer drug in several types of human cancers. Despite of several preclinical and clinical studies of curcumin, the precise mechanism of curcumin in cancer prevention has remained unclear. In our study, we for the first time investigated whole transcriptome alteration in A549 non-small cell lung cancer (NSCLC) cell lines after treatment with curcumin using RNA sequencing. We found that lots of genes and signaling pathways were significantly altered after curcumin treatment in A549 cells. With bioinformatics approaches (gene ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING), we found that those curcumin altered genes were not only the genes that induce cell death but also those extracellular matrix receptors and mitogen-activated protein kinase signaling pathway genes which regulate cell migration and proliferation. Among those significantly altered genes, eight genes ( COL1A1, COL4A1, COL5A1, LAMA5, ITGA3, ITGA2B, DDIT3, and DUSP1) were further examined by quantitative reverse transcription polymerase chain reaction and western blot analysis in four non-small cell lung cancer cell lines. Both in cell lines and in mouse model, the extracellular matrix receptors including the integrin ( ITGA3 and ITGA2B), collagen ( COL5A1), and laminin ( LAMA5) were significantly inhibited by curcumin at messenger RNA and protein levels. Functional studies confirmed that curcumin not only induced A549 cell death but also repressed cell proliferation and migration by regulating extracellular matrix receptors. Collectively, our study suggests that curcumin may be used as a promising drug candidate for intervening lung cancer in future studies.

The effect of lipoaspirates vs. dissected abdominal fat on breast cancer cells in vitro.

BACKGROUND: Cancer cells are typically surrounded by stromal cells and embedded in extracellular matrix (ECM). The stromal compartment interacts with cancer cells to promote growth and metastasis. For decades, autologous fasciocutaneous flaps have been safely applied for breast reconstruction after mastectomy. In contrast, the safety of fat grafting (lipofilling) procedure has been under debate regarding the risk of cancer recurrence. METHODS: Harvested fat tissue (lipoaspirates) and dissected abdominal fat (DAF) were co-cultured with MCF-7 breast cancer cells. The vitality of MCF-7 cells was measured using AlamarBlue® consecutively for 5 days. ECM degradation was determined by detection of matrix metalloproteinase-1 (MMP-1) expression in MCF-7 cells. Integrin α2 was measured by Western blot to assess the degree of adhesion and motility of MFC-7 cells. RESULTS: The MCF-7 proliferation increased substantially when co-cultured with fat tissue. However, there was no significant difference between the proliferation stimulating effects of lipoaspirates and DAF. Similarly, MMP-1 protein expression was equally elevated in MCF-7 cells by both lipoaspirates and DAF. Importantly, MCF-7 cells showed an increased level of integrin α2 once co-cultured with either lipoaspirates or DAF. CONCLUSION: Fat tissue increases the proliferation of MCF-7 cells in vitro. Our data suggest that lipoaspirates as well as DAF might possess a considerable potency to promote tumorigenic growth of breast cancer cells. Thus, clinical trials are needed to address the safety of lipofilling by breast reconstruction surgery after mastectomy.

The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet's Membrane Components.

PURPOSE: To clarify the adherent properties of cultured human corneal endothelial cell (cHCEC) subpopulations (SPs). METHODS: Each SP was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC SPs were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. Adhered cells were then evaluated by phase-contrast microscopy. RESULTS: The cHCECs were bound to laminin-511, laminin-411, and Type-IV collagen in a concentration-dependent manner, yet weakly bound to Perlecan, Agrin, and TSP-1. Comparison of the influence of cell-suspension vehicles on cHCEC attachment showed that cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Next, we compared the adherent properties of HCEC SPs. Both the fully differentiated, mature cHCEC SP and the epithelial-to-mesenchymal-transitioned (EMT)-phenotype SP were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those SPs. Although the level of cells adhered to the laminin-411-coated plate was the same among the cHCEC SPs, the fully differentiated, mature cHCEC SP was significantly more tightly bound to laminin-511 than was the EMT-phenotype SP. CONCLUSIONS: The findings of this study suggest that the binding ability of cHCECs to major Descemet's membrane components is distinct among cHCEC SPs, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy.

Characterization of Regulatory T-Cell Markers in CD4+ T Cells of the Upper Airway Mucosa.

CD4+ T regulatory cells (Tregs) comprise a heterogeneous population of cells the regulate immune responses and prevent autoimmunity. Most reports on human Tregs are derived from studies of peripheral blood, although Tregs mainly exert their functions in the periphery. Here we performed a detailed analysis of Tregs in the human upper airway mucosa under non-inflammatory conditions, and found that 10% of all CD4+ T cells expressed the transcription factor FOXP3 and the memory marker CD45RO, as well as high levels of CTLA-4. The majority of FOXP3+CD4+ T cells co-expressed the transcription factor Helios and produced very little cytokines, compatible with being thymus-derived Tregs. FOXP3+Helios-CD4+ T cells were more heterogeneous. A mean of 24% produced the immunomodulatory cytokine IL-10, whereas a large fraction also produced IL-2, IFN-µ or IL-17. A significant population (6%) of FOXP3-negative T cells also produced IL-10, usually in combination with IFN-µ. Together, we found that CD4+ T cells in the upper airways differed functionally from their counterparts in peripheral blood, including higher expression of IL-10. Moreover, our findings suggest that several subsets of CD4+ T cells with functionally distinct regulatory properties reside in the upper airway mucosa which should be taken into account when targeting Tregs for therapy.

Megakaryocyte- and megakaryocyte precursor-related gene therapies.

Hematopoietic stem cells (HSCs) can be safely collected from the body, genetically modified, and re-infused into a patient with the goal to express the transgene product for an individual's lifetime. Hematologic defects that can be corrected with an allogeneic bone marrow transplant can theoretically also be treated with gene replacement therapy. Because some genetic disorders affect distinct cell lineages, researchers are utilizing HSC gene transfer techniques using lineage-specific endogenous gene promoters to confine transgene expression to individual cell types (eg, ITGA2B for inherited platelet defects). HSCs appear to be an ideal target for platelet gene therapy because they can differentiate into megakaryocytes which are capable of forming several thousand anucleate platelets that circulate within blood vessels to establish hemostasis by repairing vascular injury. Platelets play an essential role in other biological processes (immune response, angiogenesis) as well as diseased states (atherosclerosis, cancer, thrombosis). Thus, recent advances in genetic manipulation of megakaryocytes could lead to new and improved therapies for treating a variety of disorders. In summary, genetic manipulation of megakaryocytes has progressed to the point where clinically relevant strategies are being developed for human trials for genetic disorders affecting platelets. Nevertheless, challenges still need to be overcome to perfect this field; therefore, strategies to increase the safety and benefit of megakaryocyte gene therapy will be discussed.

Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

Three-Dimensional Environment Sustains Hematopoietic Stem Cell Differentiation into Platelet-Producing Megakaryocytes.

Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbß3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals.

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and ß1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Evaluation of the expression of stem cell markers in human breast cancer reveals a correlation with clinical progression and metastatic disease in ductal carcinoma.

The tumor stem cell theory could explain how patients with metastatic disease show clinical relapse several months after starting treatment due to the survival of a small group of cells with unique characteristics. We examined the distribution and expression of a panel of stem cell markers in human breast cancer primary tumors. Human breast tissues were processed for immunohistochemistry, and RNA was extracted for analysis by quantitative-PCR. Immunohistochemical assay revealed that CD44 was strongly expressed in background endothelia and epithelia. CD133 expression was lost in tumor-associated endothelial cells. Conversely, CD49b was strongly stained in the tumors, associated vessels and ducts but was weakly stained in the background epithelia. q-PCR analysis revealed that CD44 and PSCA were reduced in patients with poor outcome (metastatic disease and death from breast cancer), with a marked reduction in ductal carcinoma, particularly with metastasis to bone although these did not reach significant difference. CD133 was significantly reduced in patients with metastatic disease and was also significantly reduced in patients with ductal carcinoma/bone metastasis. Conversely, CD49F was increased in patients with a poor outcome and those with ductal cancer and bone metastases. This is the first study to determine the distribution and expression pattern of these stem cell markers in human breast cancer. There was a significant association between loss of expression and metastatic disease in patients with breast cancer. Such differential expression may play a part in breast cancer disease progression, and suggests that the current stem cell theory may not hold true for all cancer types.

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins.

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and ß5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.

Overexpression of integrin a2 promotes osteogenic differentiation of hBMSCs from senile osteoporosis through the ERK pathway.

UNLABELLED: Osteoporosis is a major health problem affecting the aging population, especially in patients 65 years of age and older. The imbalance between bone formation and bone resorption is generally accepted as the essential mechanism leading to osteoporosis. In addition to the abnormal activation of osteoclast-mediated bone resorption, the dysfunction of bone marrow stromal cells (BMSCs) in mediating bone formation has been accepted as a major contributor to the progression of senile osteoporosis. RESULTS: In our study, senile osteoporotic hBMSCs displayed a decreasing capacity for proliferation and osteoblast differentiation, which was associated with the downregulation of integrin α2. Forced ectopic integrin α2 expression using a lentivirus vector reversed the dysfunction of senile osteoporotic hBMSCs. Additionally, the overexpression of integrin α2 upregulated the levels of Runx2 and Osterix. Mechanically, Western blot analyses revealed that integrin α2 phosphorylated ERK1/2 and the inactivation of ERK by PD98059 suppressed the osteoblastic differentiation of hBMSCs, suggesting that integrin α2 promotes osteoblast proliferation through the activation of ERK1/2 MAPK. CONCLUSION: Taken together, our results show that hBMSCs obtained from senile osteoporotic patients gradually lose their capability to differentiate along the osteogenic lineage and proliferate, which might be associated with the abnormal regulation of the integrin α2/ERK/Runx2 signaling pathway undergoing senile osteoporosis.

TNF-α promotes cerebral pericyte remodeling in vitro, via a switch from α1 to α2 integrins.

BACKGROUND: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. METHODS: The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). RESULTS: Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. CONCLUSIONS: Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner.

Immunoexpression of α2-integrin and Hsp47 in hereditary gingival fibromatosis and gingival fibromatosis-associated dental abnormalities.

OBJECTIVE: The purpose of the present study was to investigate the expression of the α2-integrin subunit and heat shock protein 47 (Hsp47) in two families with isolated gingival fibromatosis (GF) form and one family with GF associated with dental abnormalities and normal gingival (NG). STUDY DESIGN: Immunohistochemistry was performed with antibodies against α2-integrin and Hsp47 in specimens from two unrelated families with hereditary gingival fibromatosis (Families 1 and 2) and from one family with a gingival fibromatosis-associated dental abnormality (Family 3); NG samples were used for comparison. The results were analysed statistically. RESULTS: Immunoreactivity for α2-integrin and Hsp47 was observed in the nucleus of epithelial cells of both the basal and suprabasal layer and a more discreet signal was noted in connective tissue in all study samples. Hsp47 showed higher immunoreactivity in Family 2 compared with the other families (p ≤ 0.05). Despite the markup α2-integrin was higher in Family 3 there was no statistically significant difference between the families studied (p ≥ 0.05). CONCLUSIONS: Our results confirmed the heterogeneity of GF, such that similar patterns of expression of the condition may show differences in the expression of proteins such as Hsp47. Although no difference in α2-integrin expression was observed between GF and NG groups, future studies are necessary to determine the exact role of this protein in the various forms of GF and whether it contributes to GF pathogenesis.

Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and ß1-triggered motility, migration and invasion of prostate cancer cells.

BACKGROUND: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. METHODS: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3(par)) or resistant (PC3(res)) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and ß subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. RESULTS: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3(res). The α2 and ß1 integrin subtypes were dramatically elevated, integrins α1 and α6 were lowered, whereas α5 was nearly lost in PC3(res). Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3(par) cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α2 and ß1 integrins significantly trigger the motile behaviour of the tumour cells. CONCLUSION: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.

Stimulation of platelet apoptosis by peptidoglycan from Staphylococcus aureus 113.

Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30 min exposure to monomeric fraction (mPGN) (≥50 ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)ß(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1 µM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.

Preimplantation factor (PIF) promoting role in embryo implantation: increases endometrial integrin-α2ß3, amphiregulin and epiregulin while reducing betacellulin expression via MAPK in decidua.

BACKGROUND: Viable embryos secrete preimplantation factor (PIF), a peptide that has autocrine effects where levels correlate with cultured embryos development. sPIF (PIF synthetic analog) promotes implantation by regulating decidual-cells immunity, adhesion, apoptosis and enhances trophoblastic cell invasion. Herein sPIF priming effects on non-decidualized endometrium and decidualized-stroma are investigated, assessing elements critical for effective embryo-maternal cross-talk, prior to and at implantation. METHODS: We tested sPIF effect on human non-pregnant endometrial epithelial and non-decidualized stroma α2ß3 integrin expression (IHC and flow cytometry), comparing with scrambled PIF (PIFscr-control). We examined sPIF effect on decidualized non-pregnant human endometrial stromal cells (HESC) determining pro-inflammatory mediators expression and secretion (ELISA) and growth factors (GFs) expression (Affymetrix global gene array). We tested sPIF effect on HESC Phospho-kinases (BioPlex) and isolated kinases activity (FastKinase). RESULTS: sPIF up-regulates α2ß3 integrin expression in epithelial cells, (P < 0.05) while PIFscr had no effect. In contrast, in stromal cell cultures sPIF had no effect on the same. In HESC, sPIF up-regulates pro-inflammatory cytokines; IL8, IL1ß and IL6 expression. The major increase in GRO-α, ICAM-1 and MCP-3 expression is coupled with same ligands secretion (P < 0.05). sPIF modulates in HESC GFs expression: up-regulates amphiregulin and epiregulin- critical for implantation and enhances several fibroblast growth factors (FGF) relevant for decidual function. In contrast, sPIF down-regulates major pro-proliferative ligands, betacellulin and IGF1 expression. sPIF modulatory effect on GFs is exerted by down-regulating pro-proliferative phospho-activated MAPkinases, p-MEK1 and p-ERK (P < 0.01, P < 0.04, respectively). Stress-induced p-38-MAPK (P = 0.04) and c-Jun kinase signaling involved MAPK8IP2 (-2.1 fold) expression decreased which protects against reactive oxygen species. Although pro-inflammatory p-NFkB (P = 0.06) decrease was mild, its promoter TNFRS11 expression markedly (-25-fold) decreased. In contrast, anti-proliferative phosphatases PTPRZ1 and PPP2R2C expression increased. CONCLUSIONS: sPIF post-fertilization primes endometrial-epithelium, while during implantation creates a beneficial pro-inflammatory milieu. PIF acts by balancing decidual pro-implantation properties while controlling excessive pro-proliferative and inflammatory signals expression. Overall, PIF influences critical peri-implantation events in a sequential coordinated fashion which facilitates embryo implantation.