Implantation of medical devices can result in inflammation. A large library of polymers is screened, and a selection found to promote macrophage differentiation towards pro-or anti-inflammatory phenotypes. The bioinstructive properties of these materials are validated within a rodent model. By identifying novel materials with immune-instructive properties, the relationship between material-immune cell interactions could be investigated, and this offers exciting possibilities to design novel bioinstructive materials that can be used for numerous clinical applications including medical implants.

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion (P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin alpha(v)beta(3)-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior.

Metastatic disease is dependent on tumor cell migration through the venous and lymphatic systems and requires dynamic rearrangement of adherens junctions. Endocytosis of cadherins is a key mechanism to dynamically arrange adherens junctions, signaling, and motility in tumor cells; however, the role of shear in regulating this process in metastatic cells is unknown. In this study, the role of shear in regulating cell surface expression of E-cadherin was investigated. M etastatic cell survival is dependent on adhesion and migration under shear stress encountered in the lymphatic and venous circulation. A key mechanism in this process involves altering cell surface expression of specific integrins and cadherins. Recent work has shown that both integrins and cadherins enter into early endosomes where they can be recycled back to the plasma membrane or degraded in lysosomes.(1,2)More recently, we (3) and others (4) have shown that exposure to physiological shear rates induces activation, translocation, and clustering of integrins in tumor cells; however, the precise role for shear in modulating cadherin expression in metastatic cells is unknown.Downregulation of cadherin-based adherens junctions is an important step in epithelial tumor invasion and metastasis, (5) and can occur through transcriptional silencing, (6) protein degradation,or through endocytosis. (8) Although transcriptional repression of the E-cadherin gene can result in loss of E-cadherin expression, more recent evidence suggests that post-translational mechanisms such as endocytosis may be involved in migratory behavior. (9) Accumulating evidence suggests that whenever epithelial cells need to become motile, E-cadherin is rapidly removed from the plasma membrane, where it can be recycled to sites of new cell-cell contacts.(10,11) Recently, Palacios et al. have shown that endocytosed E-cadherin enters into early Rab5-positive endosomes where it can be recycled rapidly back to the cell surface (time scale of 15-30 min) without undergoing degradation.(11) The Src family of tyrosine kinases have been implicated in regulating this process as they can phosphorylate tyrosine residues in the short intraplasmic tail of E-cadherin, thereby promoting their internalization by endocytosis.(12) In a recent study by Avizienyte et al., elevated Src in colon cancer cells was shown to alter adhesive properties that are associated with the disorganization of E-cadherin-dependent cell-cell contacts.(13) As loss of E-cadherin is associated with cellular invasion, (14) we hypothesized that shear may alter E-cadherin expression in metastatic tumor cells (OC-1 cells) in a Src-regulated manner.In the present study, the role of shear in the trafficking of E-cadherin within OC-1 cells was investigated. We show that shear induces internalization of E-cadherin into Rab5-positive endosomes, in contrast to static OC-1 cells where E-cadherin localizes specifically to cell-cell contacts. Src inhibition, using the specific inhibitor 4-amino-5-(4-methylpheny1)-7-(t-buty1) pyrazolo [3,4...

The ubiquitin–proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPStreated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s Ϫ1 . At a venous shear rate of 400 s Ϫ1 , 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s Ϫ1 . This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s Ϫ1 . The integrin ␣v␤3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of ␣v␤3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s Ϫ1 . Collectively, our data suggest that ␣v␤3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions. platelet releasate; vitronectin; fibronectin ESOPHAGEAL CANCER IS ONE of the least studied and deadliest cancers worldwide. At the time of diagnosis, Ͼ50% of patients have radiographically visible metastases (12). Metastases arise through a complex series of sequential steps involving a variety of tumor and host properties. A key step is emigration from the circulation. Metastasis occurs primarily in the venous circulation (20). This relatively lowshear venous environment facilitates the arrest of tumor cells to the vessel wall, eventually leading to penetration of the vascular wall (21). Previous experiments suggested that cancer cell binding to endothelium has a key role in metastases; however, these experiments were performed under static conditions (32). The interaction of metastatic cells with the endothelium under fluid flow conditions is not well understood. Recent evidence suggests that tumor adherence occurring in the presence of shear stress is analogous to the manner in which platelets (6) and leukocytes tether, adhere, and roll (4).In the present investigation, we studied a shear-specific effect on metastatic cells and further characterized the interaction of tumor cells, platelets, and inflamed endothelium. Successful cell arrest in the circulation is dependent on the balance between adhesive and antiadhesive forces and on the rate at which adhesive interactions are broken (33). The init...

Parkinson’s disease (PD) is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of Lewy bodies (LB) in a proportion of the remaining neurones. α-synuclein is the main component of LB, but the pathological mechanisms that lead to neurodegeneration associated with LB formation remain unclear. Three pivotal elements have emerged in the development of PD: α-synuclein, mitochondria and protein degradation systems. We previously reported a unique model, created by conditional genetic depletion of 26S proteasomes in the SNpc of mice, which mechanistically links these three elements with the neuropathology of PD: progressive neurodegeneration and intraneuronal inclusion formation. Using this model, we tested the hypothesis that α-synuclein was essential for the formation of inclusions and neurodegeneration caused by 26S proteasomal depletion. We found that both of these processes were independent of α-synuclein. This provides an important insight into the relationship between the proteasome, α-synuclein, inclusion formation and neurodegeneration. We also show that the autophagy-lysosomal pathway is not activated in 26S proteasome-depleted neurones. This leads us to suggest that the paranuclear accumulation of mitochondria in inclusions in our model may reflect a role for the ubiquitin proteasome system in mitochondrial homeostasis and that neurodegeneration may be mediated through mitochondrial factors linked to inclusion biogenesis.

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal-glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases.

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.