Immunostaining For Fibronectin Research Articles

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Intranasal Delivery of miR133b in a NEO100-Based Formulation Induces a Healing Response in Spinal Cord-Injured Mice.

Despite important advances in the pre-clinical animal studies investigating the neuroinhibitory microenvironment at the injury site, traumatic injury to the spinal cord remains a major problem with no concrete response. Here, we examined whether (1) intranasal (IN) administration of miR133b/Ago2 can reach the injury site and achieve a therapeutic effect and (2) NEO100-based formulation of miR133b/Ago2 can improve effectiveness. 24 h after a cervical contusion, C57BL6 female mice received IN delivery of miR133b/Ago2 or miR133b/Ago2/NEO100 for 3 days, one dose per day. The pharmacokinetics of miR133b in the spinal cord lesion was determined by RT-qPCR. The role of IN delivery of miR133b on motor function was assessed by the grip strength meter (GSM) and hanging tasks. The activity of miR133b at the lesion site was established by immunostaining of fibronectin 1 (FN1), a miR133b target. We found that IN delivery of miR133b/Ago2 (1) reaches the lesion scar and co-administration of miR133b with NEO100 facilitated the cellular uptake; (2) enhanced the motor function and addition of NEO100 potentiated this effect and (3) targeted FN1 expression at the lesion scar. Our results suggest a high efficacy of IN delivery of miR133b/Ago2 to the injured spinal cord that translates to improved healing with NEO100 further potentiating this effect.

Robotic device for transcranial focussed ultrasound applications in small animal models.

Focussed Ultrasound (FUS) combined with microbubbles (MBs) was proven a promising modality for non-invasive blood brain barrier disruption (BBBD). Herein, two devices for FUS-mediated BBBD in rodents are presented. A two-axes robotic device was manufactured for navigating a single element FUS transducer of 1MHz relative to the brain of rodents. A second more compact device featuring a single motorized vertical axis was also developed. Their performance was assessed in terms of motion accuracy, MRI compatibility and trans-skull BBBD in wild type mice using MBs in synergy with pulsed FUS. Successful BBBD was evidenced by the Evans Blue dye method, as well as by Fibronectin and Fibrinogen immunostaining. BBB permeability was enhanced when the applied acoustic intensity was increased. The proposed devices constitute a cost-effective and ergonomic solution for FUS-mediated BBBD in small animal models. Further experimentation is needed to examine the repeatability of results and optimise the therapeutic protocol.

Fibroblast-like synovial cell production of extra domain A fibronectin associates with inflammation in osteoarthritis

BackgroundThe pathophysiology of osteoarthritis (OA) involves wear and tear, and a state of low-grade inflammation. Tissue repair responses include transforming growth factor beta (TGFβ)-induced myofibroblast production of extracellular matrix. Fibronectins are an essential part of the extracellular matrix, and injection of fibronectin fragments into rabbit joints is a previously established animal model of OA. Fibronectin containing the ED-A domain is currently being used as drug delivery target in the development of anti-inflammatory drugs (e.g. Dekavil).MethodsIn this study, samples of synovial membrane were obtained from patients with knee OA undergoing joint replacement surgery. Immunostaining for ED-A fibronectin and the myofibroblast marker alpha smooth muscle actin (αSMA) was performed on fibroblast-like synovial cells (FLS) and synovial membranes. RAW 264.7 macrophages were incubated with recombinant ED-A fibronectin.ResultsThe staining of ED-A fibronectin in OA FLS was increased by TGFβ but not by TNFα, lipopolysaccharide, or IL-6 (n = 3). ED-A fibronectin co-stained with the myofibroblast marker αSMA in both the OA FLS (n = 3) and in the OA synovial membranes (n = 8). ED-A fibronectin staining was associated with both number of lining layer cells (rho = 0.85 and p = 0.011) and sublining cells (rho = 0.88 and p = 0.007) in the OA synovium (n = 8), and co-distributed with TNFα (n = 5). Recombinant ED-A fibronectin increased the production of TNFα by RAW 264.7 macrophages (n = 3).ConclusionsThe disease process in OA shares features with the chronic wound healing response. Our findings support utilizing ED-A fibronectin for drug delivery or therapeutic targeting to reduce pro-inflammatory responses in OA.

492-P: A Novel Renoprotective Target of SGLT2 Inhibitor: HIF-1α Inhibition and AMPK Activation in Renal Proximal Tubular Epithelial Cells

The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None.

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and β-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of β-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and β-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The β-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the β-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of β-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena.

Evaluation of a Titanium Surface Treated with Hydroxyapatite Nanocrystals on Osteoblastic Cell Behavior: An In Vitro Study.

In the context of macrostructural and microstructural modifications to the design of dental implants, surface topography changes with different treatments have the purpose of accelerating bone formation. The aim of this study was to evaluate in vitro the influence of aggregated hydroxyapatite nanocrystals to surfaces treated with double acid etching (Nano) on osteoblastic cell behavior compared with a conventional double acid-etched surface (DE). Commercially pure Grade 4 titanium discs (6 × 2 mm) were selected, and both cell proliferation and viability were assessed at 24, 48, and 72 hours using Trypan blue vital dye and MTT, respectively. The expression of type I collagen and osteopontin on such surfaces was evaluated using ELISA. Immunostaining for fibronectin was also performed. Quantitative data were analyzed statistically using two-way analysis of variance (ANOVA) followed by Bonferroni post-test with a 5% significance level. The results showed that in all evaluated time periods, cells expressed fibronectin on both surfaces. The cells presented greater morphologic spreading on the Nano surface when compared with the conventional DE surface in all assessed times. Increased cell proliferation and viability were detected in the Nano surface (P < .05) when compared with the conventional DE surface, especially after 72 hours. Osteopontin expression was higher after 24 hours in the Nano surface when compared with the conventional DE surface (P < .05). For type I collagen, a higher expression was observed with the Nano surface than with the DE surface, again after 72 hours (P < .05). This in vitro study showed that the treated Nano surface tested promoted increased cell proliferation and viability when compared with the control surface. Additionally, increased cell spreading as well as type I collagen and osteopontin secretion were observed, favoring the early events of osseointegration.

X-Linked Alport Dogs Demonstrate Mesangial Filopodial Invasion of the Capillary Tuft as an Early Event in Glomerular Damage.

BackgroundX-linked Alport syndrome (XLAS), caused by mutations in the type IV collagen COL4A5 gene, accounts for approximately 80% of human Alport syndrome. Dogs with XLAS have a similar clinical progression. Prior studies in autosomal recessive Alport mice demonstrated early mesangial cell invasion as the source of laminin 211 in the glomerular basement membrane (GBM), leading to proinflammatory signaling. The objective of this study was to verify this process in XLAS dogs.MethodsXLAS dogs and WT littermates were monitored with serial clinicopathologic data and kidney biopsies. Biopsies were obtained at set milestones defined by the onset of microalbuminuria (MA), overt proteinuria, onset of azotemia, moderate azotemia, and euthanasia. Kidney biopsies were analyzed by histopathology, immunohistochemistry, and electron microscopy.ResultsXLAS dogs showed progressive decrease in renal function and progressive increase in interstitial fibrosis and glomerulosclerosis (based on light microscopy and immunostaining for fibronectin). The only identifiable structural abnormality at the time of microalbuminuria was ultrastructural evidence of mild segmental GBM multilamination, which was more extensive when overt proteinuria developed. Co-localization studies showed that mesangial laminin 211 and integrin α8β1 accumulated in the GBM at the onset of overt proteinuria and coincided with ultrastructural evidence of mild cellular interpositioning, consistent with invasion of the capillary loops by mesangial cell processes.ConclusionIn a large animal model, the induction of mesangial filopodial invasion of the glomerular capillary loop leading to the irregular deposition of laminin 211 is an early initiating event in Alport glomerular pathology.

Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development.

We present an experimental method allowing the production of three-dimensional organ-like structures, namely microtissues (MTs), in vitro without the need for exogenous extracellular matrix (ECM) or growth factors. Submandibular salivary glands (embryonic day ED14), kidneys (ED13) and lungs (ED13) were harvested from mouse embryos and dissociated into single cells by enzyme treatment. Single cells were seeded into special hanging drop culture plates (InSphero) and cultured for up to 14days to obtain MTs. This strategy permitted full control of the quantity of seeded cells. The development of the MTs into organs was followed histologically and immunohistochemically. Well-organized epithelial structures surrounded by a basal lamina were formed, as confirmed by transmission electron microscopy. Expression of E-cadherin, vimentin, fibronectin and α-SMA was compared in organs and corresponding MTs by real-time quantitative polymerase chain reaction. Branching morphogenesis was induced in MTs (as shown by histology and immunostaining for fibronectin and perlecan) and was conserved even after 14days of culture. MTs continued their development and their epithelial structures were comparable with those of the physiological organ at postnatal day 2 (PN2). Expression of aquaporins was investigated to obtain better support for the functional differentiation of epithelial cells. Histogenesis proceeded and led to the start of organogenesis. This experimental model might improve our knowledge of epithelial-mesenchymal histogenesis and can be employed to study development or cellular organization during the embryonic formation of organs.

On the stability of adsorbed and grafted fibronectin coatings on fluorocarbon surfaces for cardiovascular applications

Event Abstract Back to Event On the stability of adsorbed and grafted fibronectin coatings on fluorocarbon surfaces for cardiovascular applications Vanessa Montano-Machado1, 2, Ludivine Hugoni1, 2, Sergio Díaz-Rodríguez1, Ranna Tolouei1, Pascale Chevallier1, Emmanuel Pauthe2 and Diego Mantovani1 1 CHU de Quebec, Research Center, Laval University, Lab. Biomaterials & Bioengineering CRC-I, Dept. Min-Met-Mater Eng, Canada 2 University of Cergy-Pontoise, ERRMECe, France Introduction: When a dispositive is implanted in the human body its clinical success is influenced by the first interactions its surface establishes with the surrounding biological tissues and fluids. In the case of cardiovascular applications, endothelialization is still the missed element. Although numerous surfaces modifications based on biomolecules coatings have been reported in the literature, the stability of the coatings is rarely assessed for blood applications involving circulation. This study focuses on the development of a long-term-stable fibronectin (FN) coating on fluorocarbon films (CFx) able to promote endothelial-cell response on cardiovascular devices. Materials and Methods: Cleaned stainless steel substrates were first coated by a plasma deposition process, leading to CFx films[1]. Afterwards, FN was added following two approaches: adsorption for 30 min at 37 ºC (FNa) or CFx film functionalization followed by FN grafting through glutaric anhydride (FNg)[2]. Deformation tests were performed in order to simulate cardiovascular stenting procedure. Samples were tested under static and dynamic conditions during 7 days in PBS solution[3]. Coating characterizations were performed through X-Ray Photoelectron Spectroscopy, Contact Angle (CA), Atomic Force Microscopy (AFM), Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and immunostaining of FN with polyclonal and monoclonal antibodies. Haemolysis rate (40 min) and endothelial cell viability test (7 days) were completed. Results and Discussion: XPS survey analysis demonstrated the CFx deposition after plasma process, as well as the presence of both FN coatings (Table 1). Furthermore, high resolution of C1s evidenced that CF2 bonds from CFx were still detected on FNa whereas for FNg this contribution was no more detectable, but only the picks corresponding to the protein. The CA was very high for CFx film, 122.1 ± 4.2º, and it decreased to 104.2 ± 7.2º and 67.2 ± 3.6º for FNa and FNg respectively (Figure 1). These results evidenced differences in covering of the CFx film and/or in the protein conformation. However immunostaining assays showed a homogeneous coating for both FNa and FNg samples. Regarding AFM analysis, CFx film presented a Rq and Rsk values of 1 nm and 0.5 respectively, for FNa these values were 4 nm and -1.6, while for FNg were 4.5 nm and 0.6, respectively. ToF-SIMS imaging and depth profiling analysis showed denser coatings of FNg when compared to FNa. After stability tests, both FNa and FNg coatings showed high stability after the deformation process and after static tests. However, after 7 days of dynamic test, FNg presented a more homogeneous and denser coating mainly evidenced by the preservation of the CA, the chemical composition and a decrease of the Rq value. Regarding biological tests (Figure 2), significant improvement on cell viability was detected of both FNa and FNg coatings when compared to the CFx film. FNg showed better hemocompatibility properties than FNa coatings. Conclusions: Two FN coatings, either adsorbed (FNa) or grafted (FNg), have been characterized by using different techniques and submitted to several stability tests. FN grafted exhibited higher stability and denser coatings than FNa. The pertinence of FNg coating for cardiovascular applications was confirmed also by high endothelial cell viability and hemocompatibility properties. This work was partially funded by NSERC-Canada, FRQ-NT-Quebec, CFI-Canada, Québec/France Samuel de Champlain Program, Centre de Recherche sur les Matériaux Avancés CERMA-ULaval, Research Center of the CHU de Québec and Wallonie-Bruxelles International. VMM was awarded of a doctoral scholarship from CONACYT – National Council of Science and Technology, Mexico. ToF-SIMS analysis were performed by Céline Noël at the Research Centre in Physics of Matter and Radiation (PMR) in Namur University, Belgium. The authors would like to express their gratitude for their collaboration on the performance of experiments and/or their technical assistance to Laurent Houssiau, Jean-Jacques Pireaux and Stéphane Turgeon.

Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction

Event Abstract Back to Event Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction Annie Zhe Cheng1, 2, Nikolaj Gadegaard1, Matthew Dalby2* and Manuel Salmeron-Sanchez1* 1 University of Glasgow, Biomedical Engineering, United Kingdom 2 University of Glasgow, Centre for Cell Engineering, United Kingdom Introduction: The study of material-protein interactions is important in the development of biomaterials for tissue engineering. We previously showed that poly(ethyl acrylate) (PEA) induces the organisation of fibronectin (FN) into nanonetworks in a process known as material-driven fibrillogenesis[1] and exposes specific structural domains of FN for growth factor (GF) binding[2]. In addition, the incorporation of microscale features is interesting in biomaterials as it allows the mimicking of in vivo systems, whereby cells thrive in a microscale environment[3]. Here we use a simple photolithographic procedure to fabricate micropatterns of PEA, allowing material-driven fibrillogenesis of FN to occur in microscale regions. The synergistic relationship between FN nanonetworks and growth factors is also investigated with respect to mesenchymal stem cell (MSC) response, in particular adhesion, migration, and differentiation. Materials and Methods: Photolithography (PL): PEA is spin-coated onto glass substrates, which are subsequently coated with photoresist and patterned with a photomask via exposure to UV light. Oxygen plasma is used to etch PEA and expose glass for PEG-silane grafting, yielding a non-fouling background. Residual photoresist is removed to expose the underlying PEA for protein coating. Surface characterisation: Micropatterned surfaces coated with FN and BMP-2 on PEA are characterised by SEM, AFM, immunogold staining, and immunofluorescence to verify successfu protein patterning on the substrates. Cell culture: MSCs are seeded on micropatterned, PEA-modified surfaces coated with FN and/or BMP-2 for up to 4 weeks to study adhesion, migration, and differentiation. Results and Discussion: PEA is patterned on 100 μm × 100 μm square islands using PL. FN is coated onto these patterns and immunostaining of FN is carried out to visualise the resulting FN microdomains. The interaction between FN and PEA is confirmed using AFM, which reveals FN nanonetworks on the microdomains. Additionally, BMP-2 localisation is visualised by immunogold labelling. When MSCs are cultured on micropatterned, protein-modified surfaces, they selectively adhere to the FN microdomains while avoiding the non-fouling background. The osteogenic potential of these micropatterned surfaces is investigated. With the incorporation of BMP-2 within the FN nanonetworks, MSC differentiation into osteoblasts is observed on the microscale islands, indicating the ability to spatially control MSC differentiation using bioactive cues including chemical interfaces and GF signalling. Conclusions: We demonstrate a simple method of patterning microdomains of PEA using PL and subsequently coating the microdomains with FN and BMP-2 to facilitate synergistic interactions and influence MSC fate. This approach can be utilized with different shapes, dimensions, and order, and can also be downsized to yield much smaller feature sizes, enabling the precise design of bioactive surfaces to promote osteogenesis in MSCs. Medical Research Council; James Watt Nanofabrication Centre

Abstract P023: Toll-like Receptor 4 Deficiency Reduces Macrophage Mediated Renal Injury in Ang-II Induced Hypertension

Infiltration of macrophages has been amply demonstrated in the kidney of humans and experimental models of hypertension however, its role in tissue injury and repair remains unclear. Furthermore, recent studies implicate upregulation of toll-like receptor 4 (TLR4) and inflammation in hypertension and vascular remodeling. Previously, we observed that C3H/HeJ mice lacking functional TLR4 were hyporesponsive to Ang-II induced hypertension and renal damage. In this study, we hypothesized that TLR4 deficiency inhibits macrophage mediated inflammation and TGF-β-induced fibroblast accumulation to reduce renovascular injury and fibrosis. Male C3H/HeOuJ (functional TLR4) and C3H/HeJ mice (dysfunctional TLR4) aged 8-10 weeks underwent subcutaneous osmotic pump insertion for infusion of Angiotensin-II (Ang-II) at 1000 ng/kg/min or vehicle (0.9% saline) for 4 weeks. Blood pressure (BP) was measured twice weekly by non-invasive tail-cuff plethysmography. Ang-II treatment increased mean BP in both groups but to a greater degree in C3H/HeOuJ mice compared to C3H/HeJ (138.6± 6.3 vs. 103.4±7.2 mm/Hg). Plasma creatinine in saline treated C3H/HeOuJ and C3H/HeJ mice was normal (0.37±0.026 and 0.32±0.023 mg/dL respectively) in contrast to ang-II treated mice (1.34±0.075 vs. 0.78±0.052 mg/dL). Flow cytometry revealed increased F4/80+ and CD40+ cells indicating pro-inflammatory M1 macrophage in Ang-II treated C3H/HeOuJ compared to C3H/HeJ mice. Similarly, Ang-II increased the mRNA and protein expression of CD40, MCP-1, TNF-α and TGF-β in C3H/HeOuJ compared to C3H/HeJ mice. Immunostaining revealed increased colocalization of CD45 and procollagen-1 in renal sections of C3H/HeOuJ mice suggesting increased accumulation of myeloid fibroblasts than in C3H/HeJ mice. The expression of α-smooth muscle actin an indicator of myofibroblast and extracellular matrix proteins, collagen type I and fibronectin (Western blot and immunostaining) was increased in C3H/HeOuJ compared to C3H/HeJ mice. In conclusion, our data suggests that in C3H/HeJ mice, deficiency of functional TLR4 reduces macrophage activation and inflammation which in turn decreases TGF-β-induced renal fibrotic response in Ang-II induced hypertension.

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression.

BackgroundComparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors.Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts.MethodsIn vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored.ResultsWhile normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells.ConclusionsOur results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.

Cholinergic differentiation of neural stem cells generated from cell aggregates-derived from Human Bone marrow stromal cells

Human Bone marrow stromal cells (hBMSCs) can differentiate under appropriate experimental conditions into neuronal and glial-like cells. This study shows a protocol for producing human neural stem cells (hNSCs) from hBMSCs and the subsequent differentiation of hNSCs into cholinergic neurons (CNs), where sequential media replaced the culturing media. hBMSCs have been used in generating cell aggregates (CAs) using bFGF, EGF and B27. The hNSCs were isolated from CAs, and the CNs differentiated from the hNSCs using sequential media, where bFGF, EGF and B27 were gradually replaced with NGF. The hNSC stemness was checked by RT-PCR of SOX2, Oct-4 and Nanog genes. Fibronectin, CD90, CD106, CD31, nestin, neurofilament 68 (NF-68), NF-200 and ChAT immunostaining evaluated the differentiation of the hBMSCs, the hNSCs and the CNs. FM1-43 was used in studying the function of the CNs. The hBMSCs were immunoreactive to fibronectin, CD90 and CD106; they were checked for lipogenic and osteogenic differentiation. The cells of the CAs were immunoreactive to nestin. The hNSCs were immunoreactive to nestin and NF-68, also, they expressed SOX2, Oct-4 and nanong. Nestin expression declined sharply following NSC differentiation into CNs, while the expression of NF-200, synapsin I, synaptophysin, MAP-2 and ChAT increased. They were stained with FM1-43, where the synaptic vesicles were released following stimulation. The present study demonstrates the conversion of hBMSCs into CASs under appropriate conditions. CAs generated hNSCs, which were induced in order to differentiate into CNs using sequential media, where the yield was 83%.

Abstract 129: AIP1 suppresses tumor metastasis by regulating tumor microenvironment and metastatic niche

Abstract AIP1 (also called DAB2IP, a new member of RAS-GAP family protein) is downregulated in a variety of human cancer tissues. We and others have recently reported that AIP1 functions as a tumor suppressor in prostate cancer models. However, the role of AIP1 in regulating tumor microenvironment and metastatic niche is not known. In the present study, we implanted tumor cells with the same genetic background into C57BL/6 wild type (WT) and AIP1-deficient (AIP1-KO) mice to address this question. In subcutaneous (SQ) allograft models using B16 melanoma and LLC Lewis lung carcinoma, tumor growth and metastasis to multiple organs including lymph node, lung, liver, spleen and bone marrow were significantly enhanced in AIP1-KO host, suggesting AIP1 in tumor microenvironment limits tumor metastases. However, a direct intravenously injection of tumor cells resulted in no differences in tumor metastasis between WT and AIP1-KO mice, indicating AIP1 in host regulates tumor metastasis at steps prior to extravasation to target tissues. We examined effects of AIP1 deletion on the induction of local inflammation, tumor cell epithelial-mesenchymal transition (EMT), generation of metastatic routes (formation of new blood and lymphatic vessels) and establishment of pre-metastatic niche in distance tissues. SQ tumors in AIP1-KO host showed more profound increases in macrophage infiltration, tumor cell EMT, intratumoral angiogenesis and peri-tumoral lymphangiogenesis as well as lymphangiogenesis in lymph nodes prior to the detection of metastatic tumor cells. Finally, SQ tumor induced an establishment of pre-metastatic niche in lung as indicated by increased immunostaining for fibronectin and accumulation of VEGFR1, c-Kit and CXCR-positive cells which were augmented in AIP1-KO mice. Importantly, WT recipient with AIP1-KO bone marrow-derived cells showed similar phenotype to AIP1-KO mice in tumor growth and metastasis. Taken together, our data suggest that AIP1 functions as tumor metastasis suppressor by regulating tumor microenvironment, metastatic routes and pre-metastatic niche. Citation Format: Wang Min. AIP1 suppresses tumor metastasis by regulating tumor microenvironment and metastatic niche. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 129. doi:10.1158/1538-7445.AM2014-129

Maintenance of undifferentiated state of human induced pluripotent stem cells through cytoskeleton-driven force acting to secreted fibronectin on a dendrimer-immobilized surface

Understanding of the fundamental mechanisms that govern adhesive properties of human induced pluripotent stem cells (hiPSCs) to culture environments provides surface design strategies for maintaining their undifferentiated state during cell expansion. Polyamidoamine dendrimer surface with first-generation (G1) with dendron structure was used for co-cultures of hiPSCs and SNL feeder cells that formed tightly packed compact hiPSC colonies, similar to those on a conventional gelatin-coated surface. hiPSCs passaged up to 10 times on the G1 surface maintained their undifferentiated state. Immunostaining and reverse transcriptase PCR analysis of fibronectin showed that the secreted fibronectin matrix from feeder cells on the G1 surface contributed to hiPSC attachment. Compared with cells on the gelatin-coated surface, F-actin and paxillin immunostaining revealed a well-organized network of actin stress fibers and focal adhesion formation at cell-substrate sites in hiPSC colonies on the G1 surface. E-cadherin expression levels on these surfaces were almost same, but paxillin and Rac1 expression levels on the G1 surface were significantly higher than those on the gelatin-coated surface. Zyxin showed prominent expression on the G1 surface at sites of focal adhesion and cell-cell contact in colonies, whereas zyxin expression on the gelatin-coated surface was not observed in regions of cell-cell contact. These findings indicate that transduction of mechanical stimuli through actin polymerization at sites of focal adhesion and cell-cell contact results in maintenance of undifferentiated hiPSC colonies on G1 surface. The G1 surface enables a substrate design based on the mechanical cues in the microenvironment from feeder cells to expand undifferentiated hiPSCs in long-term culture.

β-Phase poly(vinylidene fluoride) films encouraged more homogeneous cell distribution and more significant deposition of fibronectin towards the cell–material interface compared to α-phase poly(vinylidene fluoride) films

The piezoelectric response from β-phase poly(vinylidene fluoride) (PVDF) can potentially be exploited for biomedical application. We hypothesized that α and β-phase PVDF exert direct but different influence on cellular behavior. α- and β-phase PVDF films were synthesized through solution casting and characterized with FT-IR, XRD, AFM and PFM to ensure successful fabrication of α and β-phase PVDF films. Cellular evaluation with L929 mouse fibroblasts over one-week was conducted with AlamarBlue® metabolic assay and PicoGreen® proliferation assay. Immunostaining of fibronectin investigated the extent and distribution of extracellular matrix deposition. Image saliency analysis quantified differences in cellular distribution on the PVDF films. Our results showed that β-phase PVDF films with the largest area expressing piezoelectric effect elicited highest cell metabolic activity at day 3 of culture. Increased fibronectin adsorption towards the cell–material interface was shown on β-phase PVDF films. Image saliency analysis showed that fibroblasts on β-phase PVDF films were more homogeneously distributed than on α-phase PVDF films. Taken collectively, the different molecular packing of α and β-phase PVDF resulted in differing physical properties of films, which in turn induced differences in cellular behaviors. Further analysis of how α and β-phase PVDF may evoke specific cellular behavior to suit particular application will be intriguing.

Reduction of Aldosterone Production Improves Renal Oxidative Stress and Fibrosis in Diabetic Rats

Aldosterone is increased in diabetes and contributes to the development of diabetic nephropathy. The authors hypothesized that reduction in aldosterone production in diabetes by amlodipine or aliskiren improves diabetic kidney disease by attenuating renal oxidative stress and fibrosis. Normoglycemic and streptozotocin-induced diabetic Sprague-Dawley rats were given vehicle, amlodipine, or aliskiren alone and combined for 6 weeks. At the end of study, we evaluated blood pressure (BP), 24-hour urinary sodium (UNaV) and aldosterone excretion rates, renal interstitial fluid (RIF) levels of nitric oxide (NO), cyclic guanosine 3',5'-monophosphate (cGMP), and 8-isoprostane, and renal morphology. BP was not significantly different between any of experimental groups. UNaV increased in diabetic animals and was not affected by different treatments. Urinary aldosterone excretion increased in diabetic rats receiving vehicle and decreased with amlodipine and aliskiren alone or combined. RIF NO and cGMP levels were reduced in vehicle-treated diabetic rats and increased with amlodipine or aliskiren given alone and combined. RIF 8-isoprostane levels and renal immunostaining for periodic acid-Schiff and fibronectin were increased in vehicle-treated diabetic rats and decreased with aliskiren alone or combined with amlodipine. The authors conclude that inhibition of aldosterone by amlodipine or aliskiren ameliorates diabetes induced renal injury via improvement of NO-cGMP pathway and reduction in oxidative stress and fibrosis, independent of BP changes.

Muscle Stretching after Immobilization Applied at Alternate Days Preserves Components of Articular Cartilage

We compared the response of articular cartilage subjected to muscle stretching at different frequencies after joint immobilization. Wistar rats with immobilized left hind limbs were classified into the following groups: immobilization, immobilization followed by muscle stretching applied daily (group IS7) or three times a week (IS3), muscle stretching applied daily (S7) or three times a week (S3), and a control group (C) that underwent no intervention. We then evaluated the cartilage for cellularity, loss of proteoglycans, collagen density, and immunostaining of fibronectin and chondroitin 4-sulfate. Group IS7 showed a significant increase in cellularity and significant loss of proteoglycan compared with the control. In addition, IS7 group had less proteoglycan than IS3. Thin collagen fibrils were significantly reduced in IS7 rats, compared with IS3 and C. There was a significant decrease in the amount of thick fibrils in all groups compared with the control. Groups IS7 and IS3 showed significantly more intense fibronectin immunostaining than the other groups. Our results show that if applied daily after immobilization, muscle stretching is harmful to articular cartilage. However, when applied on alternate days, muscle stretching preserves the components of articular cartilage. We suggest that the latter frequency is more suitable for treatment.

Diagnostic value of E-cadherin and fibronectin in differentiation between reactive mesothelial and adenocarcinoma cells in serous effusions

Background:One of the problems in studying serous effusion cytological samples is differentiation of reactive mesothelial cells from metastatic adenocarcinoma cells.Materials and Methods:In this study, the immunohistochemical diagnostic value of E-cadherin and fibronectin markers for differentiation of these 2 groups of cells was studied. 50 cell block samples prepared from serous effusions were examined. Based on clinical and histological studies, 25 cases had primary carcinoma, and the other 25 were proved to be benign effusion cases. All the cases were studied for E-cadherin and fibronectin immunostaining using an envision technique. Statistical analyzes were performed employing Chi-square and exact Fisher tests, using SPSS software (version 16).Results:24 of the 25 benign cases were stained with fibronectin and 2 with E-cadherin, whereas from among the 25 metastatic cases, 2 reacted to fibronectin and 22 to E-cadherin. Considering the staining of the 2 markers under conditions that the cells were stained with fibronectin but not with E-cadherin, positive predictive value (PPV) and negative predictive value (NPV) to identify reactive mesothelial cells were 100% and 92.5% while under conditions that had not been stained with fibronectin but with E-cadherin, PPV and NPV to detect adenocarcinoma cells were 95.2% and 82.1%, respectively.Conclusion:Employing this short panel can be helpful for better differentiation of adenocarcinoma and reactive mesothelial cells in serous fluids.