Losartan Slows Pancreatic Tumor Progression and Extends Survival of SPARC-Null Mice by Abrogating Aberrant TGFβ Activation

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

<p>Abstract</p> <p>Background</p> <p>Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.</p> <p>Methods</p> <p>A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured <it>in vitro </it>and <it>in vivo </it>in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific <it>in situ </it>hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.</p> <p>Results</p> <p>The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. <it>In vitro </it>and <it>in vivo </it>(subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels <it>in vitro </it>and <it>in vivo</it>, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the <it>in vitro </it>cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific <it>in situ </it>hybridization.</p> <p>Conclusions</p> <p>The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.</p

Matrix Development in Self-Assembly of Articular Cartilage

Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs

SPARC: a matricellular regulator of tumorigenesis

Although many clinical studies have found a correlation of SPARC expression with malignant progression and patient survival, the mechanisms for SPARC function in tumorigenesis and metastasis remain elusive. The activity of SPARC is context- and cell-type-dependent, which is highlighted by the fact that SPARC has shown seemingly contradictory effects on tumor progression in both clinical correlative studies and in animal models. The capacity of SPARC to dictate tumorigenic phenotype has been attributed to its effects on the bioavailability and signaling of integrins and growth factors/chemokines. These molecular pathways contribute to many physiological events affecting malignant progression, including extracellular matrix remodeling, angiogenesis, immune modulation and metastasis. Given that SPARC is credited with such varied activities, this review presents a comprehensive account of the divergent effects of SPARC in human cancers and mouse models, as well as a description of the potential mechanisms by which SPARC mediates these effects. We aim to provide insight into how a matricellular protein such as SPARC might generate paradoxical, yet relevant, tumor outcomes in order to unify an apparently incongruent collection of scientific literature

Substrate counterdiffusion and reaction in membrane-attached biofilms: mathematical analysis of rate limiting mechanisms

A mechanistic model of organic substrate biodegradation in membrane-attached biofilms growing in extractive membrane bioreactors is presented and analysed to establish the rate-limiting steps. The model accounts for counterdiffusion and reaction of oxygen and organic substrate within the biofilm, and predicts detailed substrate concentration profiles and the evolution over time of biofilm thickness. Good agreement was found between model predictions and organic substrate flux and biofilm thickness measured experimentally in a lab-scale single-tube extractive membrane bioreactor. Analysis using this model showed that, due to oxygen diffusion limitations, the reaction zone within the biofilm is located at the biofilm/biomedium boundary and constitutes a small fraction of the entire biofilm volume. This allows a considerable simplification of biofilm modelling. A simple diffusion model was formulated as an alternative to the more complex full diffusion-reaction model for the calculation of organic substrate flux. This simple model is based on the insight that the organic compound flux is limited primarily by the biofilm diffusion resistance. The diffusion model was combined to a yield-based expression for biofilm accumulation to give the evolution over time of biofilm thickness. The simplified model predicts, as accurately as the full mechanistic model, the biofilm thickness and organic substrate flux. (C) 1999 Elsevier Science Ltd. All rights reserved

Modelling and analysis of membrane-attached biofilms

The theory of diffusion and reaction has been applied to describe mass transfer and reaction phenomena in membrane-attached biofilms (MABs) growing in extractive membrane bioreactors (EMBs), and to establish the rate-limiting mechanisms in these systems. The model formulated accounts for substrate counter-diffusion and two-limiting-substrate reaction within the biofilm. Model simulations are compared to experimental data obtained in a lab-scale EMB and a simple case study is considered to show how MABs affect performance of EMBs. It is found that the organic substrate flux across the membrane is strongly affected by MABs, which constitute an additional resistance to mass transfer and in most cases reduce the flux across the membrane. As a result of the investigation, it is concluded that the decrease of flux in the presence of MABs, observed experimentally and predicted theoretically, is dominated by the resistance to organic substrate transfer caused by the biofilm

TLR3 activation modulates immunomodulatory properties of human periodontal ligament cells

Background: Toll-like receptors (TLR) are a group of receptors that play roles in the innate immune system. Human periodontal ligament cells (hPDL cells) express several TLRs, including TLR3, a nucleotide sensing receptor that recognizes double-stranded RNA from viral infection. However, its role in hPDL cells is unclear. The aim of this study was to investigate the responses of hPDL cells in terms of immunomodulation after TLR3 engagement. Methods: HPDL cells were treated with various doses of poly I:C, a TLR3 activator. The expression of interferon-gamma (IFNγ), indoleamine 2,3 dioxygenase (IDO), and human leukocyte antigen G (HLA-G) was determined. Chemical inhibitors and small interfering RNA (siRNA) were used to confirm the role of TLR3. Coculture with human peripheral blood mononuclear cells (PBMCs) with poly I:C-activated hPDL cells was performed. Results: Endosomal TLR3 in hPDL cells was observed by immunocytochemistry. Addition of poly I:C significantly enhanced the expression and secretion of IFNγ, IDO, and HLA-G. Knockdown of TLR3 using siRNA decreased the poly I:C-induced expression of these three molecules. Bafilomycin-A, an inhibitor of auto-phagosome and lysosome fusion, inhibited poly I:C-induced IDO and HLA-G expression, whereas cycloheximide and a TLR3-neutralizing antibody had no effect. In co-culture experiments, poly I:C-activated hPDL cells inhibited PBMCs proliferation and increased mRNA expression of forkhead box P3 (FOXP3), a transcription factor which is a marker of regulatory T cells. Conclusion: Our findings indicated that TLR3 engagement of hPDL cells induced immunosuppressive properties of these cells. Because immunosuppressive properties play an important role in tissue healing and regeneration, activation of TLR3 may help to attenuate tissue destruction by limiting the inflammatory process and perhaps initiate the healing and regeneration process of the periodontium