Anne Koehler

Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation

A mild strain induced by matrix remodeling mechanically primes latent TGF-β1 for its subsequent activation and release in response to contractile forces.

Integrins αvβ5 and αvβ3 promote latent TGF-β1 activation by human cardiac fibroblast contraction

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.

MicroRNA-21 preserves the fibrotic mechanical memory of mesenchymal stem cells

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.

Molecular evolution of SPARC: absence of the acidic module and expression in the endoderm of the starlet sea anemone, Nematostella vectensis

The matricellular glycoprotein SPARC is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals: a Ca2+-binding glutamic acid-rich acidic domain at the N-terminus (domain I), a follistatin-like module (domain II), and an extracellular Ca2+-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 Ca2+-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 Ca2+-ions to the two EF-hands in the EC domain. We propose that further Ca2+-dependent activities emerged with the acquisition of an acidic N-terminal module in triplobastic organisms.

Plasma fibronectin stabilizes Borrelia burgdorferi–endothelial interactions under vascular shear stress by a catch-bond mechanism

Significance Spread of bacteria via the bloodstream to vital organs causes most mortality due to bacterial infection. To exit the bloodstream and enter these organs, bacteria must be able to resist the forces generated by flowing blood so that they can adhere to the endothelial cells lining blood vessels without being washed away. This process is not yet understood for most disease-causing bacteria. Here, we show that the Lyme disease pathogen Borrelia burgdorferi exploits an abundant constituent of blood, plasma fibronectin, to form endothelial interactions that become stronger as forces due to blood-flow increase. The ability to recruit this highly conserved molecule may also be important for the vascular interaction mechanisms of other pathogens. Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi. Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial–endothelial interaction biomechanics. We found that B. burgdorferi does not primarily target insoluble matrix Fn deposited on endothelial surfaces but, instead, recruits and induces polymerization of soluble plasma Fn (pFn), an abundant protein in blood plasma that is normally soluble and nonadhesive. Under physiological shear stress, caps of polymerized pFn at bacterial poles formed part of mechanically loaded adhesion complexes, and pFn strengthened and stabilized interactions by a catch-bond mechanism. These results show that B. burgdorferi can transform a ubiquitous but normally nonadhesive blood constituent to increase the efficiency, strength, and stability of bacterial interactions with vascular surfaces. Similar mechanisms may promote dissemination of other Fn-binding pathogens.

ENCAPSULATION OF MYXOBOLUS PENDULA (MYXOSPORIDIA) BY EPITHELIOID CELLS OF ITS CYPRINID HOST SEMOTILUS ATROMACULATUS

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.

ENVIRONMENTAL FACTORS AFFECTING THE DISTRIBUTION AND ABUNDANCE OF CYST-FORMING MYXOBOLUS SPP. AND THEIR CYPRINID HOSTS IN 3 LAKES IN ALGONQUIN PARK, ONTARIO

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.

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

Abstract 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.

The fibronectin ED-A domain enhances recruitment of latent TGF-β-binding protein-1 to the fibroblast matrix

ABSTRACT Dysregulated secretion and extracellular activation of TGF-β1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-β-binding protein-1 (LTBP-1) and thus stores TGF-β1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1–fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-β1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-β1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin. Highlighted Article: The presence of the ED-A splice domain enhances the ability of fibronectin to bind latent pro-fibrotic TGF-β1 and thus supports activation of myofibroblasts, which are main drivers of fibrosis.

Trichonosema algonquinensis n. sp. (Phylum Microsporidia) in Pectinatella magnifica (Bryozoa: Phylactolaemata) from Algonquin Park, Ontario, Canada

Abstract 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 × 4.4 ± 0.1 μm. 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.