F-actin Cytoskeleton Organization Research Articles

SSPH I, A Novel Anti-cancer Saponin, Inhibits EMT and Invasion and Migration of NSCLC by Suppressing MAPK/ERK1/2 and PI3K/AKT/ mTOR Signaling Pathways.

Saponin of Schizocapsa plantaginea Hance I (SSPH I).a bioactive saponin found in Schizocapsa plantaginea, exhibits significant anti-proliferation and antimetastasis in lung cancer. To explore the anti-metastatic effects of SSPH I on non-small cell lung cancer (NSCLC) with emphasis on epithelial-mesenchymal transition (EMT) both in vitro and in vivo. The effects of SSPH I at the concentrations of 0, 0.875,1.75, and 3.5 μM on A549 and PC9 lung cancer cells were evaluated using colony formation assay, CCK-8 assay, transwell assay and wound-healing assay. The actin cytoskeleton reorganization of PC9 and A549 cells was detected using the FITC-phalloidin fluorescence staining assay. The proteins related to EMT (N-cadherin, E-cadherin and vimentin), p- PI3K, p- AKT, p- mTOR and p- ERK1/2 were detected by Western blotting. A mouse model of lung cancer metastasis was established by utilizing 95-D cells, and the mice were treated with SSPH I by gavage. The results suggested that SSPH I significantly inhibited the migration and invasion of NSCLC cells under a non-cytotoxic concentration. Furthermore, SSPH I at a non-toxic concentration of 0.875 μM inhibited F-actin cytoskeleton organization. Importantly, attenuation of EMT was observed in A549 cells with upregulation in the expression of epithelial cell marker E-cadherin and downregulation of the mesenchymal cell markers vimentin as well as Ncadherin. Mechanistic studies revealed that SSPH I inhibited MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways. SSPH I inhibited EMT, migration, and invasion of NSCLC cells by suppressing MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways, suggesting that the natural compound SSPH I could be used for inhibiting metastasis of NSCLC.

Extracellular matrix stiffness modulates the mechanophenotypes and focal adhesions of colon cancer cells leading to their invasions via YAP1

Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer. It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors in vivo. However, the role of extracellular matrix (ECM) stiffness in colon cancer cell (CCCs) invasion and metastasis remains unclear. Here, bioinformatical analysis suggested that a high expression level of yes associated protein 1 (YAP1) was significantly associated with metastasis and poor prognosis in colon cancer patients. We further investigated the effects of polyacrylamide hydrogels with different stiffnesses (3, 20, and 38 ​kPa), which were simulated as ECM, on the mechanophenotype (F-actin cytoskeleton organization, electrophoretic rate, membrane fluidity, and Young's modulus) of CCCs. The results showed that a stiffer ECM could induce the maturation of focal adhesions and formation of stress fibers in CCCs, regulate their mechanophenotypes, and promote cell motility. We also demonstrated that the expression levels of YAP1 and paxillin were positively correlated in patients with colon cancer. YAP1 knockdown reduces paxillin clustering and cell motility and alters the cellular mechanophenotypes of CCCs. This is of great significance for an in-depth understanding of the invasion and metastatic mechanisms of colon cancer and for the optimization of clinical therapy from the perspective of mechanobiology.

Scinderin promotes glioma cell migration and invasion via remodeling actin cytoskeleton.

Glioma is one of the most common intracranial tumors, characterized by invasive growth and poor prognosis. Actin cytoskeletal rearrangement is an essential event of tumor cell migration. The actin dynamics-related protein scinderin (SCIN) has been reported to be closely related to tumor cell migration and invasion in several cancers. To investigate the role and mechanism of SCIN in glioma. The expression and clinical significance of SCIN in glioma were analyzed based on public databases. SCIN expression was examined using real-time quantitative polymerase chain reaction and Western blotting. Gene silencing was performed using short hairpin RNA transfection. Cell viability, migration, and invasion were assessed using cell counting kit 8 assay, wound healing, and Matrigel invasion assays, respectively. F-actin cytoskeleton organization was assessed using F-actin staining. SCIN expression was significantly elevated in glioma, and high levels of SCIN were associated with advanced tumor grade and wild-type isocitrate dehydrogenase. Furthermore, SCIN-deficient cells exhibited decreased proliferation, migration, and invasion in U87 and U251 cells. Moreover, knockdown of SCIN inhibited the RhoA/focal adhesion kinase (FAK) signaling to promote F-actin depolymerization in U87 and U251 cells. SCIN modulates the actin cytoskeleton via activating RhoA/FAK signaling, thereby promoting the migration and invasion of glioma cells. This study identified the cancer-promoting effect of SCIN and provided a potential therapeutic target for the treatment of glioma.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Investigating the mechanism of Xian-ling-lian-xia-fang for inhibiting vasculogenic mimicry in triple negative breast cancer via blocking VEGF/MMPs pathway

BackgroundXian-ling-lian-xia-fang (XLLXF), a Chinese medicine decoction, is widely used in the treatment of triple negative breast cancer (TNBC). However, the underlying mechanism of XLLXF in TNBC treatment has not been totally elucidated.MethodsHere, network pharmacology and molecular docking were used to explore the mechanism of Traditional Chinese medicine in the treatment of TNBC. Then, biological experiments were integrated to verify the results of network pharmacology.ResultsNetwork pharmacology showed that the candidate active ingredients mainly included quercetin, kaempferol, stigmasterol, and β-sitosterol through the “XLLXF–active ingredients–targets” network. Vascular endothelial growth factor A (VEGFA) and matrix metalloproteinase (MMP) 2 were the potential therapeutic targets obtained through the protein–protein interaction (PPI) network. Molecular docking confirmed that quercetin, kaempferol, stigmasterol, and β-sitosterol could stably combine with VEGFA and MMP2. Experimental verification showed that XLLXF could inhibit proliferation, colony ability, and vasculogenic mimicry (VM) formation and promote cell apoptosis in TNBC. Laser confocal microscopy found that XLLXF impaired F-actin cytoskeleton organization and inhibited epithelial mesenchymal transition. Animal experiments also found that XLLXF could inhibit tumor growth and VM formation in TNBC xenograft model. Western blot analysis and immunohistochemical staining showed that XLLXF inhibited the protein expression of VEGFA, MMP2, MMP9, Vimentin, VE-cadherin, and Twist1 and increased that of E-cadherin, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-3 in vitro and in vivo.ConclusionsIntegrating the analysis of network pharmacology and experimental validation revealed that XLLXF could inhibit VM formation via downregulating the VEGF/MMPs signaling pathway.

MiR-195 regulates intestinal epithelial restitution after wounding by altering actin-related protein-2 translation.

Early gut epithelial restitution reseals superficial wounds after acute injury, but the exact mechanism underlying this rapid mucosal repair remains largely unknown. MicroRNA-195 (miR-195) is highly expressed in the gut epithelium and involved in many aspects of mucosal pathobiology. Actin-related proteins (ARPs) are key components essential for stimulation of actin polymerization and regulate cell motility. Here, we reported that miR-195 modulates early intestinal epithelial restitution by altering ARP-2 expression at the translation level. miR-195 directly interacted with the ARP-2 mRNA, and ectopically expressed miR-195 decreased ARP-2 protein without effect on its mRNA content. In contrast, miR-195 silencing by transfection with anti-miR-195 oligo increased ARP-2 expression. Decreased ARP-2 levels by miR-195 overexpression were associated with an inhibition of early epithelial restitution, as indicated by a decrease in cell migration over the wounded area. Elevation of cellular ARP-2 levels by transfection with its transgene restored cell migration after wounding in cells overexpressing miR-195. Polyamines were found to decrease miR-195 abundance and enhanced ARP-2 translation, thus promoting epithelial restitution after wounding. Moreover, increasing the levels of miR-195 disrupted F-actin cytoskeleton organization, which was prevented by ARP2 overexpression. These results indicate that miR-195 inhibits early epithelial restitution by decreasing ARP-2 translation and that miR-195 expression is negatively regulated by cellular polyamines.

Effects of 660-nm and 780-nm Laser Therapy on ST88-14 Schwann Cells.

The aim of the present study was to evaluate the comparative effects of red (660-nm) and near-infrared (780-nm) low-level laser therapy (LLLT) on viability, mitochondrial activity, morphology and gene expression of growth factors on Schwann cells (SC). ST88-14 cells were grown in RPMI 1640 with 10mM of HEPES, 2mM of glutamine, 10% fetal bovine serum and 1% antibiotic-antimycotic solution at 37°C in humidified atmosphere of 5% CO2 . Cells were detached with trypsin and centrifugated at 231g for 5min at 10°C, and the pellet (8×104 cells/tube) was irradiated at the bottom of 50ml polypropylene tube with a Twin-Laser system (660 and 780nm, 40mW, 1mWcm-2 , 3.2 and 6.4J, 80 and 160Jcm-2 with 80 and 160s). After 1, 3 and 7days, the analysis was performed. After irradiation, the SC increase mitochondrial activity, gene expression of the neural growth factors NGF and BDNF, and cell migration and increase the G2/M cells. SC showed neuronal morphology, normal F-actin cytoskeleton organization and positive labeling for S100. PBM increased metabolic activity, mitosis and gene expression when irradiated with red and infrared LLLT. An increase in cell migration was obtained when irradiated with infrared LLLT.

Ascofuranone suppresses invasion and F-actin cytoskeleton organization in cancer cells by inhibiting the mTOR complex 1 signaling pathway.

Ascofuranone is an antiviral antibiotic that is known to exert multiple anti-tumor effects, including cell cycle arrest, inhibition of mitochondrial respiration, and inhibition of angiogenesis. In this study, we investigated the molecular mechanisms underlying the anti-metastatic effects of ascofuranone in insulin-like growth factor-I (IGF-1)-responsive cancer cells. The inhibitory effect of ascofuranone on cancer cell migration and invasion was assessed using scratch wound healing and Matrigel invasion assays, respectively. F-actin cytoskeleton organization was assessed using FITC conjugated phalloidin staining. Target gene expression was evaluated using Western blotting and gene silencing was performed using siRNA transfections. Finally, the anti-metastatic effect of ascofuranone was investigated in vivo. We found that ascofuranone suppressed IGF-1-induced cell migration, invasion and motility in multiple cancer cell lines. The effects of ascofuranone on actin cytoskeleton organization were found to be mediated by suppression of the mTOR/p70S6K/4EBP1 pathway. Ascofuranone inhibited IGF-1-induced mTOR phosphorylation and actin cytoskeleton organization via upregulation of AMPK and downregulation of Akt phosphorylation. It also selectively suppressed the IGF-1-induced mTOR complex (mTORC)1 by phosphorylation of Raptor, but did not affect mTORC2. Furthermore, we found that focal adhesion kinase (FAK) activation decreased in response to ascofuranone, rapamycin, compound C and wortmannin treatment. Finally, we found that ascofuranone suppressed phosphorylation of FAK and mTOR and dephosphorylation of Raptor in cancerous metastatic lung tissues in vivo. Our data indicate that ascofuranone suppresses IGF-1-induced cancer cell migration and invasion by blocking actin cytoskeleton organization and FAK activation through inhibition of the mTORC1 pathway, and reveal a novel anti-metastatic function of this compound.

Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells.

Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC50 of 91.4 and 109.46µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55-3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.

Influence of apple phytochemicals in ZnO nanoparticles formation, photoluminescence and biocompatibility for biomedical applications

The new green-synthesised ZnO nanoparticles (NPs) using apple (var. Starking) phytochemicals present a great potential for bioimaging applications. These NPs, when compared with ZnO microparticles synthesised with pure phytochemicals (quercetin or sucrose), and water, revealed that sizes and shapes were widely dependent on the organic precursors used. Based on these findings, new insights into the synthesis of ZnO NPs using apple phytochemicals were presented.The photoluminescent properties, characterized by steady-state and time resolve photoluminescence measurements, revealed that besides the intense sharp near-UV band edge emission observed for all particles, with sub-nanosecond lifetime, a strong broad emission band peak at 2.20 eV was detected for microparticles, with longer decay times being associate to crystal defects. Additionally, the photoluminescent properties of ZnO particles, further explored by fluorescence lifetime imaging microscopy (FLIM), suggested adequacy for imaging applications. The cytotoxicity, evaluated in human dermal fibroblasts, proving the biosafety of ZnO NPs by an unaffected cellular viability, total mitochondrial activity and F-actin cytoskeleton organization, contrasted with some degree of cytotoxicity depicted for microparticles. The influence of the phytochemicals in ZnO cytotoxicity was discussed.To the authors' best knowledge this is the first report of ZnO NPs synthesised with apple extracts. The novelty of choosing a fruit widely used in the food industry will render affordable NPs through the concept of circular economy. The proved biosafety of these ZnO NPs together with their intrinsic photoluminescent properties, open perspectives for the development of cost-effective bioimaging materials with potential to be further directed into biomedical applications.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-β1.

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

The RhoGAP Stard13 controls insulin secretion through F-actin remodeling

ObjectiveActin cytoskeleton remodeling is necessary for glucose-stimulated insulin secretion in pancreatic β-cells. A mechanistic understanding of actin dynamics in the islet is paramount to a better comprehension of β-cell dysfunction in diabetes. Here, we investigate the Rho GTPase regulator Stard13 and its role in F-actin cytoskeleton organization and islet function in adult mice. MethodsWe used Lifeact-EGFP transgenic animals to visualize actin cytoskeleton organization and dynamics in vivo in the mouse islets. Furthermore, we applied this model to study actin cytoskeleton and insulin secretion in mutant mice deleted for Stard13 selectively in pancreatic cells. We isolated transgenic islets for 3D-imaging and perifusion studies to measure insulin secretion dynamics. In parallel, we performed histological and morphometric analyses of the pancreas and used in vivo approaches to study glucose metabolism in the mouse. ResultsIn this study, we provide the first genetic evidence that Stard13 regulates insulin secretion in response to glucose. Postnatally, Stard13 expression became restricted to the mouse pancreatic islets. We showed that Stard13 deletion results in a marked increase in actin polymerization in islet cells, which is accompanied by severe reduction of insulin secretion in perifusion experiments. Consistently, Stard13-deleted mice displayed impaired glucose tolerance and reduced glucose-stimulated insulin secretion. ConclusionsTaken together, our results suggest a previously unappreciated role for the RhoGAP protein Stard13 in the interplay between actin cytoskeletal remodeling and insulin secretion.

Multifunctional PLLA-ceramic fiber membranes for bone regeneration applications

A novel method to process electrospun poly(l-lactic acid) (PLLA) membranes incorporating glass reinforced hydroxyapatite granules (gHA) interspacially between the polymeric fibers is reported, thus increasing the surface area for cellular interactions. gHA granules (≤150μm) electrospun together with the polymer solution, lead to an average fiber diameter of 550±150nm for pristine PLLA and 440±170nm for the composite samples. An increase of the overall porosity was observed, from 79±3% for the PLLA up to 88±5% for the hybrid samples, keeping material’s wettability and mechanical properties. Bone-bonding ability showed that both samples induced HA crystal nucleation, but with a distinct pattern of mineral deposition. gHA microcomposite allows a better F-actin cytoskeleton organization during the initial adhesion and spreading, favoring cell-fibers and cell-to-cell interactions and enhanced alkaline phosphatase activity, making them potential candidates for bone healing strategies.

Abstract 5090: Targeting metastatic prostate cancer with polyisoprenylated cysteinyl amides

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) poses a major clinical management challenge due to lack of effective therapies. Monomeric G- proteins of the Ras superfamily such as Rho, Rac and Cdc42 are critical mediators in prostate cancer progression and metastasis, due to their effect on F-actin organization in cell migration. These proteins are polyisoprenylated and subject to regulation by polyisoprenylated methylated protein methyl esterase (PMPMEase). We hypothesized that metabolism of monomeric G-proteins by PMPMEase regulates growth, survival and differentiation as well as cell migration and invasion through its effects on F-actin assembly. Methods: The effects of polyisoprenylated cysteinyl amides (PCAIs), NSL-RD-036, NSL-BA-040, NSL-BA-055 and NSL-BA-056 on the viability and proliferation of prostate cancer cell lines were investigated using the MTS assay. Docetaxel and paclitaxel were used as positive controls. Metastatic PC -3 cell migration and invasion in the presence of NSL-RD-036 were determined using the scratch and matrigel invasion assays. The effect of NSL-RD-040 on F-actin organization in TagRFP F-actin marker-transfected PC 3 cells was also investigated. Results: The PCAIs were cytotoxic to all prostate cancer cell lines (PC-3, DU 145, LNCaP and 22Rv1). In all the cell lines, NSL-BA-040 showed the highest activity (EC50 4.3 - 5.0 μM) followed by NSL-RD-036 (EC50 2.0 - 9.2 μM), NSL-BA-056 (EC50 4.8 - 7.5 μM) and NSL-BA-055 (EC50 2.9 - 11.2 μM) while docetaxel and paclitaxel were not as effective (EC50 >100 μM). Metastatic PC-3 cell proliferation was inhibited in a concentration-dependent manner by NSL-RD-036 and at 2 μM PC-3 cell proliferation was halted. Exposure of PC-3 cells to NSL-BA-040 induced apoptosis and disrupted F-actin cytoskeleton organization in TagRFP F-actin marker transfected metastatic PC-3 cells. NSL-RD-036 also significantly inhibited PC-3 cell migration and invasion through matrigel. Conclusions: Polyisoprenylated small molecules suppress migration and invasion as well as disrupt F-actin organization of metastatic PC-3 cells. Polyisoprenylated small molecules could serve as a new class of therapeutic agents in metastatic castration resistant prostate cancer. Citation Format: Rosemary Aniwaa Poku, Augustine Nkembo, Olufisayo Salako, Felix Amissah, Hernan Flores-Rozas, Tryphon Mazu, Nazarius S. Lamango. Targeting metastatic prostate cancer with polyisoprenylated cysteinyl amides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5090. doi:10.1158/1538-7445.AM2015-5090

Mechanical characterization of human mesenchymal stem cells subjected to cyclic uniaxial strain and TGF-β1

Human mesenchymal stem cells (hMSCs) have shown promising potential in the field of regenerative medicine particularly in vascular tissue engineering. Optimal growing of MSCs into specific lineage requires a thorough understanding of the role of mechanobiology in MSC metabolism. Although effects of external physical cues (mechanical stimuli through external loading and scaffold properties) on regulation of MSC differentiation into Smooth muscle (SM) lineage have attracted widespread attention, fewer studies are available on mechanical characterization of single engineered MSCs which is vital in tissue development through proper mechanotransductive cell–environment interactions. In this study, we investigated effects of uniaxial tensile strain and transforming growth factor-β1 (TGF-β1) stimulations on mechanical properties of engineered MSCs and their F-actin cytoskeleton organization. Micropipette aspiration technique was used to measure mechanical properties of MSCs including mean Young׳s modulus (E) and the parameters of standard linear viscoelastic model. Compared to control samples, MSCs treated by uniaxial strain either with or without TGF-β1 indicated significant increases in E value and considerable drop in creep compliance curve, while samples treated by TGF-β1 alone met significant decreases in E value and considerable rise in creep compliance curve. Among treated samples, uniaxial tensile strain accompanied by TGF-β1 stimulation not only caused higher stimulation in MSC differentiation towards SM phenotype at transcriptional level, but also created more structural integrity in MSCs due to formation of thick bundled F-actin fibers. Results can be applied in engineering of MSCs towards functional target cells and consequently tissue development.

Visceral adipose tissue and leptin increase colonic epithelial tight junction permeabilityviaa RhoA‐ROCK‐dependent pathway

Proinflammatory cytokines produced by immune cells play a central role in the increased intestinal epithelial permeability during inflammation. Expansion of visceral adipose tissue (VAT) is currently considered a consequence of intestinal inflammation. Whether VAT per se plays a role in early modifications of intestinal barrier remains unknown. The aim of this study was to demonstrate the direct role of adipocytes in regulating paracellular permeability of colonic epithelial cells (CECs). We show in adult rats born with intrauterine growth retardation, a model of VAT hypertrophy, and in rats with VAT graft on the colon, that colonic permeability was increased without any inflammation. This effect was associated with altered expression of tight junction (TJ) proteins occludin and ZO-1. In coculture experiments, adipocytes decreased transepithelial resistance (TER) of Caco-2 CECs and induced a disorganization of ZO-1 on TJs. Intraperitoneal administration of leptin to lean rats increased colonic epithelial permeability and altered ZO-1 expression and organization. Treatment of HT29-19A CECs with leptin, but not adiponectin, dose-dependently decreased TER and altered TJ and F-actin cytoskeleton organization through a RhoA-ROCK-dependent pathway. Our data show that adipocytes and leptin directly alter TJ function in CECs and suggest that VAT could impair colonic epithelial barrier.

Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling

SummaryMature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors); and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP) receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

Effect of BMP-2 from matrices of different stiffnesses for the modulation of stem cell fate

Stem cells cultured on extracellular matrix (ECM) with different stiffnesses have been shown to engage into different lineage commitments. However, in vivo, the components of the ECM are known to bind and strongly interact with growth factors. The effect, on the stem cell fate, of the cooperation between the mechanical properties and the growth factor in the same microenvironment has not yet been investigated. Here, we propose a protocol for mimicking this stem cell microenvironment with an in vitro system. This system consists in grafting (without using a spacer) biomolecules that contain N-termini groups onto hydrogel (poly(acrylamide-co-acrylic acid)) surfaces of various stiffnesses ranging from 0.5 to 70 kPa. First, we demonstrate that the commitment of mesenchymal stem cell populations changes in response to the substrate's rigidity, with myogenic differentiation occurring at 13–17 kPa and osteogenic differentiation at 45–49 kPa. Chemical grafting of soft and stiff matrices with an osteogenic factor (BMP-2mimetic peptide) results only in osteogenic differentiation. Also, when grafted on even softer gels (0.5–3.5 kPa), the BMP-2mimetic peptide had no effect on the stem cell differentiation. We prove that correct organization of F-actin cytoskeleton due to the mechanical properties of the microenvironment is necessary for BMP-induced smad1/5/8 phosphorylation and nuclear translocation. These results suggest that stem cell differentiation is dictated mechanically, but in the presence of a biochemical factor, the effect of the mechanical factor on stem cell commitment is modified. This can explain the diversity of stem cell behaviors in vivo where different growth factors are sequestrated on the ECM.

Relevance of the sterilization-induced effects on the properties of different hydroxyapatite nanoparticles and assessment of the osteoblastic cell response

Hydroxyapatite (Hap) is a calcium phosphate with a chemical formula that closely resembles that of the mineral constituents found in hard tissues, thereby explaining its natural biocompatibility and wide biomedical use. Nanostructured Hap materials appear to present a good performance in bone tissue applications because of their ability to mimic the dimensions of bone components. However, bone cell response to individual nanoparticles and/or nanoparticle aggregates lost from these materials is largely unknown and shows great variability. This work addresses the preparation and characterization of two different Hap nanoparticles and their interaction with osteoblastic cells. Hap particles were produced by a wet chemical synthesis (WCS) at 37°C and by hydrothermal synthesis (HS) at 180°C. As the ultimate in vivo applications require a sterilization step, the synthesized particles were characterized 'as prepared' and after sterilization (autoclaving, 120°C, 20 min). WCS and HS particles differ in their morphological (size and shape) and physicochemical properties. The sterilization modified markedly the shape, size and aggregation state of WCS nanoparticles. Both particles were readily internalized by osteoblastic cells by endocytosis, and showed a low intracellular dissolution rate. Concentrations of WCS and HS particles less than 500 μg ml(-1) did not affect cell proliferation, F-actin cytoskeleton organization and apoptosis rate and increased the gene expression of alkaline phosphatase and BMP-2. The two particles presented some differences in the elicited cell response. In conclusion, WCS and HS particles might exhibit an interesting profile for bone tissue applications. Results suggest the relevance of a proper particle characterization, and the interest of an individual nanoparticle targeted research.

Ethylacetate extract of Aquilaria crassna preserve actin cytoskeleton on simulated ischemia induced cardiac cell death

This paper investigates the effect of ethylacetate extract of Aquilaria crassna Pierre ex Lecomte on simulated ischemia-induced cytoskeleton disorganization in cardiac myoblast cell line, so as to provide some evidence for its traditional use. Rat cardiac myoblast cell line (H9c2), were subjected to 2 h simulated ischemia and 24 h of reperfusion, in the presence and absence of the ethylacetate extract of A. crassna. The cellular viability was determined by the reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by mitochondrial reductases. Cells were pre-treated with 5 mg/ml of A. crassna extract for 1 h before, or at the beginning of 40 min simulated ischemia. The F-actin cytoskeleton organization was assessed in cells treated with 5 mg/ml prior to simulated ischemia. Cells were fixed and stained for F-actin by phalloidin-conjugatedtetramethylrhodamine isothiocyanate (TRITC) and observed under fluorescence microscopy. The results showed that 2 h of simulated ischemia significantly produced cellular injury and cell death, which was significantly inhibited when treated with 5 mg/ml of the ethylacetate extract of A. crassna (p < 0.05). Treatment of the ethylacetate extract of A. crassna significantly preserved actin cytoskeleton organization. In conclusion, the ethylacetate extract of A. crassna reduced ischemia induced cell death preserve cytoskeleton organization. Key words: Aquilaria crassna, ischemic heart disease, myocardial ischemia, actin cytoskeleton.