Unai Silvan

Advanced glycation end-products: Mechanics of aged collagen from molecule to tissue.

Concurrent with a progressive loss of regenerative capacity, connective tissue aging is characterized by a progressive accumulation of Advanced Glycation End-products (AGEs). Besides being part of the typical aging process, type II diabetics are particularly affected by AGE accumulation due to abnormally high levels of systemic glucose that increases the glycation rate of long-lived proteins such as collagen. Although AGEs are associated with a wide range of clinical disorders, the mechanisms by which AGEs contribute to connective tissue disease in aging and diabetes are still poorly understood. The present study harnesses advanced multiscale imaging techniques to characterize a widely employed in vitro model of ribose induced collagen aging and further benchmarks these data against experiments on native human tissues from donors of different age. These efforts yield unprecedented insight into the mechanical changes in collagen tissues across hierarchical scales from molecular, to fiber, to tissue-levels. We observed a linear increase in molecular spacing (from 1.45nm to 1.5nm) and a decrease in the D-period length (from 67.5nm to 67.1nm) in aged tissues, both using the ribose model of in vitro glycation and in native human probes. Multiscale mechanical analysis of in vitro glycated tendons strongly suggests that AGEs reduce tissue viscoelasticity by severely limiting fiber-fiber and fibril-fibril sliding. This study lays an important foundation for interpreting the functional and biological effects of AGEs in collagen connective tissues, by exploiting experimental models of AGEs crosslinking and benchmarking them for the first time against endogenous AGEs in native tissue.

The spermatogonial stem cell niche in testicular germ cell tumors

Spermatogonial stem cells (SSCs) are pluripotent elements found in the adult seminiferous epithelium between Sertoli cells and a basal lamina which covers the multilayered external wall of peritubular myoid cells. The microenvironment of this pluripotent stem cell niche creates the complex and dynamic system that is necessary for the initiation of spermatogenesis, but this system also contains factors which can potentially collaborate in the progression of testicular germ cell tumors (TGCTs). In this review, we summarize our current knowledge about some important structural and molecular features related to the SSC niche, including growth factors, adhesion molecules, extracellular matrix, mechanical stress and vascularization. We discuss their possible collaborative effects on the generation and progression of TGCTs, which are a type of cancer representing the most frequent neoplasia among young men and whose incidence has grown very quickly during the past decades in North America and Europe. In this regard, a better understanding of the pluripotent stem cell niche where these malignancies arise will provide further insights into the origin of TGCTs and the mechanisms underlying their growth and invasion of adjacent and distant tissues.

High-resolution traction force microscopy on small focal adhesions - improved accuracy through optimal marker distribution and optical flow tracking

The accurate determination of cellular forces using Traction Force Microscopy at increasingly small focal attachments to the extracellular environment presents an important yet substantial technical challenge. In these measurements, uncertainty regarding accuracy is prominent since experimental calibration frameworks at this size scale are fraught with errors – denying a gold standard against which accuracy of TFM methods can be judged. Therefore, we have developed a simulation platform for generating synthetic traction images that can be used as a benchmark to quantify the influence of critical experimental parameters and the associated errors. Using this approach, we show that TFM accuracy can be improved >35% compared to the standard approach by placing fluorescent beads as densely and closely as possible to the site of applied traction. Moreover, we use the platform to test tracking algorithms based on optical flow that measure deformation directly at the beads and show that these can dramatically outperform classical particle image velocimetry algorithms in terms of noise sensitivity and error. We then report how optimized experimental and numerical strategy can improve traction map accuracy, and further provide the best available benchmark to date for defining practical limits to TFM accuracy as a function of focal adhesion size.

The role of microenvironment in testicular germ cell tumors

Testicular germ cell tumors (TGCTs) are the most frequent malignancies in adolescents and young adults. The incidence of TGCTs has doubled over the last few decades and the mechanisms underlying their pervasive growth are still poorly understood. Among them, seminomatous and non-seminomatous tumors have carcinoma in situ of the testis (CIS) as a common precursor lesion. It is currently accepted that the acquisition of genetic alterations and/or exposure to environmental factors are involved in the transition from CIS to invasive tumors. Nevertheless, although several TGCT-associated genetic aberrations have been identified, the mechanisms mediating their effects on TGCT development are still largely unknown. The aim of this review is to analyze the potential role of testicular microenvironmental factors, such as hypoxia and stroma cell-derived factors, in the acquisition by TGCT cells of an aggressive phenotype and the importance of these factors as potential therapeutic targets.

Surface-Driven Collagen Self-Assembly Affects Early Osteogenic Stem Cell Signaling.

This study reports how extracellular matrix (ECM) ligand self-assembly on biomaterial surfaces and the resulting nanoscale architecture can drive stem cell behavior. To isolate the biological effects of surface wettability on protein deposition, folding, and ligand activity, a polydimethylsiloxane (PDMS)-based platform was developed and characterized with the ability to tune wettability of elastomeric substrates with otherwise equivalent topology, ligand loading, and mechanical properties. Using this platform, markedly different assembly of covalently bound type I collagen monomers was observed depending on wettability, with hydrophobic substrates yielding a relatively rough layer of collagen aggregates compared to a smooth collagen layer on more hydrophilic substrates. Cellular and molecular investigations with human bone marrow stromal cells revealed higher osteogenic differentiation and upregulation of focal adhesion-related components on the resulting smooth collagen layer coated substrates. The initial collagen assembly driven by the PDMS surface directly affected α1β1 integrin/discoidin domain receptor 1 signaling, activation of the extracellular signal-regulated kinase/mitogen activated protein kinase pathway, and ultimately markers of osteogenic stem cell differentiation. We demonstrate for the first time that surface-driven ligand assembly on material surfaces, even on materials with otherwise identical starting topographies and mechanical properties, can dominate the biomaterial surface-driven cell response.

Distinct actin oligomers modulate differently the activity of actin nucleators

Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene‐bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so‐called lower actin dimer (LD) characterized by a Cys374‐Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin‐nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin‐related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1–3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1–3, but not of the mDia‐FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia‐FH2 domains, gelsolin and G1–3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits.

Evidence for a role of matrix metalloproteinases and their inhibitors in primordial germ cell migration

Understanding the mechanisms that enable migrating cells to reach their targets is of vital importance, as several pathologies, including cardiac defects and some tumours, are consequences of altered cell migration. With a view to evaluating if matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in the active migration of primordial germ cells (PGCs) from their place of origin in extra‐embryonic sites towards their final destination in the developing gonads, we analysed the expression of mRNAs encoding nine MMPs and four TIMPs in migrating (E10.5) and post‐migrating (E12.5) PGCs by means of quantitative polymerase chain reaction and the presence of MT1‐MMP in the membrane of these cells. Our results show that PGCs express MMP‐2, MMP‐9, MMP‐11, MT1‐MMP, TIMP‐1, TIMP‐2 and TIMP‐3 at both migrating and non‐migrating stages. Comparing expression levels of MMP genes between E10.5 and E12.5 PGCs revealed higher expression in migrating PGCs of MT1‐ MMP (10.3‐fold), MMP‐2 (4.8‐fold), MMP‐11 (3.2‐fold) and MMP‐9 (2.1‐fold). Similarly, the levels of TIMP gene expression were always higher in E12.5 genital ridge somatic cells: TIMP‐3 (3.4‐fold), TIMP‐1 (2.4‐fold) and TIMP‐2 (1.8‐fold). Moreover, the analysis at protein level showed the presence of MT1‐MMP in the membrane of migrating PGCs whereas the expression of these metalloproteinase is not detected once the PGCs have reach the urogenital ridges and stop migrating. These results suggest that the change from the motile to non‐motile phenotype that occurs during PGC maturation to gonocytes may be mediated in part by enhanced expression of MMPs in migrating PGCs together with higher expression of TIMPs in E12.5 genital ridges.

Actin ADP‐ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F‐actin

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3‐intoxicated HeLa cells transfected with AcGFP‐actin shows condensation of F‐actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F‐actin stress fibres. Recombinant TccC3 toxin ADP‐ribosylates purified skeletal and non‐muscle actin at threonine148 leading to a strong propensity to polymerize and F‐actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148‐ADP‐ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1–3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP‐ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148‐ADP‐ribosylated actin to curled F‐actin bundles even in the presence of thymosin β4, gelsolin or G1–3. Thr148‐ADP‐ribosylated F‐actin cannot be depolymerized by gelsolin or G1–3 as verified by TIRF, co‐sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin‐1.

Embryonic and Cancer Stem Cells - two views of the same landscape

According to the Cancer Stem Cell (CSC) theory, there is a small subset of neoplastic cells in the tumors, which retain unlimited proliferative capacity. These cells give also rise to a differentiated cancer progeny that does not have spreading potential but makes up the bulk of the tumor. Thus, CSCs would be the ultimate cause of tumor growth, maintenance and recurrence (Figure 1). CSCs are experimentally defined by the ability to recapitulate the heterogeneity of the original tumor when transplanted into immunocompatible or nude mice. In 1867 Julius Cohnheim proposed that tumors are derived from embryonal cells that rest in the adult tissues. Later on, in the middle of the following century, Furth and Kanh (1937) and Pierce and Dixon (1959) proved the stem cell properties of a subset of cells in leukemia and testicular germ cell tumors (TGCTs) and Till and McCulloch (1961) transplanted colony forming units (CFU) from the bone marrow into lethally irradiated mice. Additionally, the group of Barry Pierce showed the in vitro modulation of cancer cell differentiation (Pierce & Verney, 1961) and the in vivo cloning of single embryonal carcinoma (EC) cells, proving their pluripotency (Kleinsmith & Pierce, 1964). The differentiation of cloned leukemic cells (Pluznik & Sachs, 1965) and the reprogramming of embryonal carcinoma (EC) cells when injected into early embryos (Brinster, 1974) were also outstanding results. All these discoveries paved the way for the isolation of embryonic stem (ES) cells (Evans and Kaufman, 1981; Martin, 1981) and one of the above groups also studied the differentiation of CSCs from distinct tumors, like squamous cell carcinoma, chondrosarcoma and adenocarcinoma (Pierce & Wallace, 1971; Pierce, 1974; Pierce et al., 1977) seeding the concept of cancer differentiation therapy (Pierce & Speers, 1988). A new progress in this matter was done with the isolation of CSCs in human acute myeloid leukemia (Lapidot et al., 1994) and, particularly, with the discovery of new markers for progenitor cells in several solid tumors, such as breast (Al-Hajj et al., 2003), brain (Singh et al., 2003) and colon (O’Brien et al., 2007) cancer. During the evolution of CSC research, there have been several technical improvements that have undoubtedly contributed to the success in the engraftment of the tumor cells in the host mice and the subsequent tumor formation. Firstly, it has been proved that the immune system of the host notably affects the survival of the transplanted cells, and thus, the use of

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon-a novel ex vivo mouse explant model: MINIMAL MECHANICAL LOAD AND TENDON CULTURE CONDITIONS

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well‐characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load‐deprived environment or in a custom‐designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum‐free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load‐deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative‐like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low‐magnitude mechanical loading. Thus a minimal explant culture model featuring serum‐free medium with low mechanical loads seems to provide a useful foundation for further investigations.