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The fibrotic encapsulation of implants involves the mechanical activation of myofibroblasts and of pro-fibrotic transforming growth factor beta 1 (TGF-β1). Here, we show that both softening of the implant surfaces and inhibition of the activation of TGF-β1 reduce the fibrotic encapsulation of subcutaneous silicone implants in mice. Conventionally stiff silicones (elastic modulus, ~2 MPa) coated with a soft silicone layer (elastic modulus, ~2 kPa) reduced collagen deposition as well as myofibroblast activation without affecting the numbers of macrophages and their polarization states. Instead, fibroblasts around stiff implants exhibited enhanced intracellular stress, increased the recruitment of αv and β1 integrins, and activated TGF-β1 signalling. In vitro, the recruitment of αv integrin to focal adhesions and the activation of β1 integrin and of TGF-β were higher in myofibroblasts grown on latency-associated peptide (LAP)-coated stiff silicones than on soft silicones. Antagonizing αv integrin binding to LAP through the small-molecule inhibitor CWHM-12 suppressed active TGF-β signalling, myofibroblast activation and the fibrotic encapsulation of stiff subcutaneous implants in mice. The fibrotic encapsulation of subcutaneous silicone implants in mice can be suppressed by softening the implant surface or inhibiting the integrin-mediated activation of transforming growth factor beta 1.

Aims Pathological tissue remodelling by myofibroblast contraction is a hallmark of cardiac fibrosis. Myofibroblasts differentiate from cardiac fibroblasts under the action of transforming growth factor-β1 (TGF-β1), which is secreted into the extracellular matrix as a large latent complex. Integrin-mediated traction forces activate TGF-β1 by inducing a conformational change in the latent complex. The mesenchymal integrins αvβ5 and αvβ3 are expressed in the heart, but their role in the activation of TGF-β1 remains elusive. Here, we test whether targeting αvβ5 and αvβ3 integrins reduces latent TGF-β1 activation by cardiac fibroblasts with the goal to prevent the formation of α-smooth muscle actin (α-SMA)-expressing cardiac myofibroblasts and their contribution to fibrosis. Methods and results Using a porcine model of induced right ventricular fibrosis and pro-fibrotic culture conditions, we show that integrins αvβ5 and αvβ3 are up-regulated in myofibroblast-enriched fibrotic lesions and differentiated cultured human cardiac myofibroblasts. Both integrins autonomously contribute to latent TGF-β1 activation and myofibroblast differentiation, as demonstrated by function-blocking peptides and antibodies. Acute blocking of both integrins leads to significantly reduced TGF-β1 activation by cardiac fibroblast contraction and loss of α-SMA expression, which is restored by adding active TGF-β1. Manipulating integrin protein levels in overexpression and shRNA experiments reveals that both integrins can compensate for each other with respect to TGF-β1 activation and induction of α-SMA expression. Conclusions Integrins αvβ5 and αvβ3 both control myofibroblast differentiation by activating latent TGF-β1. Pharmacological targeting of mesenchymal integrins is a possible strategy to selectively block TGF-β1 activation by cardiac myofibroblasts and progression of fibrosis in the heart.

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide–resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

Expansion on stiff culture substrates activates pro-fibrotic cell programs that are retained by mechanical memory. Here, we show that priming on physiologically soft silicone substrates suppresses fibrogenesis and desensitizes mesenchymal stem cells (MSCs) against subsequent mechanical activation in vitro and in vivo, and identify the microRNA miR-21 as a long-term memory keeper of the fibrogenic program in MSCs. During stiff priming, miR-21 levels were gradually increased by continued regulation through the acutely mechanosensitive myocardin-related transcription factor-A (MRTF-A/MLK-1) and remained high over 2 weeks after removal of the mechanical stimulus. Knocking down miR-21 once by the end of the stiff-priming period was sufficient to erase the mechanical memory and sensitize MSCs to subsequent exposure to soft substrates. Soft priming and erasing mechanical memory following cell culture expansion protects MSCs from fibrogenesis in the host wound environment and increases the chances for success of MSC therapy in tissue-repair applications.

Spores of Myxobolus pendula develop within the cores of complex cysts on the gill arch of creek chub Semotilus atromaculatus. Adjacent to, and surrounding, the spores are concentric layers of stratified interdigitating cells, whose nature was examined by transmission electron microscopy and by immunohistochemical and molecular biological techniques. In situ hybridization data using parasite-derived ribosomal DNA as a probe indicate that infection leads to the encapsulation of developing plasmodia by host immune cells that form an epithelioid granuloma. Epithelioid cell-cell adhesion is effected by desmosomes anchored intracellularly to cytokeratin intermediate filaments. High levels of alkaline phosphatase activity are associated with regions of cellular interdigitation. The granuloma may serve to limit the spread of the parasite to surrounding tissues but does not appear to inhibit diffusion of oxygen and nutrients to the developing spores.

A mild strain induced by matrix remodeling mechanically primes latent TGF-β1 for its subsequent activation and release in response to contractile forces., Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.

Fibroblasts differentiate into the highly synthetic and contractile myofibroblast phenotype when exposed to substrates with an elastic modulus corresponding to pathologically stiff fibrotic tissue. Cellular responses to changes in substrate stiffness are typically analyzed after hours or days, which does not enable the monitoring of myofibroblast persistence, a hallmark of fibrosis. To determine long-lasting effects on the fibrotic behavior of lung fibroblasts, we followed a novel approach of explanting and repeatedly passaging fibroblasts on silicone substrates with stiffness representing various states of lung health. Fibrotic activity was determined by assaying for myofibroblast proliferation, cell contractility, expression of α-smooth muscle actin, extracellular matrix and active TGFβ1. As predicted, myofibroblast activity was low on healthy soft substrates and increased with increasing substrate stiffness. However, explanting and mechanically priming lung fibroblasts for 3 weeks on pathologically stiff substrates resulted in sustained myofibroblast activity even after the cells were returned to healthy soft cultures for 2 weeks. Such primed cells retained higher fibrotic activity than cells that had been exclusively cultured on soft substrates, and were not statistically different from cells continuously passaged on stiff surfaces. Inversely, priming lung fibroblasts for 3 weeks on soft substrates partially protected from myofibroblast activation after the shift to stiff substrates. Hence, mechano-sensed information relating to physical conditions of the local cellular environment could permanently induce fibrotic behavior of lung fibroblasts. This priming effect has important implications for the progression and persistence of aggressive fibrotic diseases such as idiopathic pulmonary fibrosis.

A lack of host-derived SPARC promotes disease progression in an intraperitoneal (IP) ID8 mouse model of epithelial ovarian cancer (EOC). Since orthotopic injection (OT) of ID8 cells better recapitulates high-grade serous cancer, we examined the impact of host-derived SPARC following OT injection. Sparc(-/-) and wild-type (WT) mice were injected with ID8 cells either OT or IP and tumors were analyzed at the moribund stage. Sparc(-/-) mice had reduced survival and fewer well-defined abdominal lesions compared with WT controls after IP injection, whereas no differences were observed in survival or abdominal lesions between Sparc(-/-) and WT mice after OT injection. No differences in mass or collagen content were observed in ovarian tumors between OT-injected Sparc(-/-) and WT mice. The abdominal wall of the IP-injected Sparc(-/-) mice exhibited immature and less abundant collagen fibrils compared with WT mice both in injected and non-injected controls. In contrast to human EOC, SPARC was expressed by the tumor cells but was absent in reactive stroma of WT mice. Exposure to the ovarian microenvironment through OT injections alters the metastatic behaviour of ID8 cells, which is not affected by the absence of host-derived SPARC.

SummaryDipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono-and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k 12/k 21 decreased when the number of PEG chains coupled to dipetarudin increased. It means that the intercompartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

The matricellular glycoprotein SPARC is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals: a Ca(2+)-binding glutamic acid-rich acidic domain at the N-terminus (domain I), a follistatin-like module (domain II), and an extracellular Ca(2+)-binding (EC) module that contains two EF-hands and two collagen-binding epitopes (domain III). We report that four SPARC orthologs (designated nvSPARC1-4) are expressed by the genome of the starlet anemone Nematostella vectensis, a diploblastic basal cnidarian composed of an ectoderm and endoderm separated by collagen-based mesoglea. We also report that domain I is absent from all N. vectensis SPARC orthologs. In situ hybridization data indicate that N. vectensis SPARC mRNAs are restricted to the endoderm during post-gastrula development. The absence of the Ca(2+)-binding N-terminal domain in cnidarians and conservation of collagen-binding epitopes suggests that SPARC first evolved as a collagen-binding matricellular glycoprotein, an interaction likely to be dependent on the binding of Ca(2+)-ions to the two EF-hands in the EC domain. We propose that further Ca(2+)-dependent activities emerged with the acquisition of an acidic N-terminal module in triplobastic organisms.

A new species of microsporidian, Trichonosema algonquinensis, is described from a freshwater bryozoan, Pectinatella magnifica from Ontario, Canada. The parasite develops in epithelial cells and appears as white, spherical masses throughout the tissues. Trichonosema algonquinensis is diplokaryotic, diploblastic and undergoes development in direct contact with the cytoplasm of the host cell. Mature spores are ovoid, tapered at one end, and measure 8.5 +/- 0.3 x 4.4 +/- 0.1 microm. The polar filament is wound in 20 to 23 helical coils. Although the parasite resembles T. pectinatellae described from the same host in Michigan and Ohio, it differs in the length of the spore and number of coils of the polar filament. Analysis of 16S rDNA by maximum likelihood, parsimony and Baysian inference, complements the morphological data in supporting the placement of T. algonquinensis as a sister species of T. pectinatellae.

In 1999, 4 species of cyprinid were surveyed for myxozoan parasites in a watershed in Algonquin Park, Canada; Kathlyn Lake, Broadwing Lake, and Lake Sasajewun were included. Eight species of Myxobolus were found that differed in their prevalence and distribution among the 3 lakes. The oligochaetes and environmental parameters, including sediment types and aquatic plants, of these 3 lakes were surveyed the following year. Oligochaetes belonging to 17 species were collected from the 3 lakes. The distribution patterns of the oligochaete fauna, with respect to the environmental variables, were examined using canonical correspondence analysis. Naidids were predominant in all 3 lakes, particularly in the pebbly and sandy sediment of Lake Sasajewun. The highest percentage of tubificids occurred in the detritus and muddy substrate of Broadwing Lake. These findings indicate that the prevalence of certain oligochaetes is congruent with the absence or presence of particular myxozoan species and that substrates and aquatic plants influence the distribution of certain oligochaete species.