Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion.

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreERT2:R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification.

Macrophages Guard Endothelial Lineage by Hindering Endothelial-to-Mesenchymal Transition: Implications for the Pathogenesis of Systemic Sclerosis.

The signals that control endothelial plasticity in inflamed tissues have only been partially characterized. For example, it has been shown that inadequate vasculogenesis in systemic sclerosis (SSc) has been associated with an endothelial defect. We used a genetic lineage tracing model to investigate whether endothelial cells die or change phenotypically after fibrosis induction and whether signals released by cells of the innate immune system and in the blood of patients influence their commitment. We observed that in the lineage-tracing transgenic mice Cdh5-CreERT2::R26R-EYFP, endothelial-derived cells (EdCs) underwent fibrosis after treatment with bleomycin, and EdCs retrieved from the lung showed expression of endothelial-to-mesenchymal transition (EndoMT) markers. Liposome-encapsulated clodronate was used to assess macrophage impact on EdCs. Clodronate treatment affected the number of alternatively activated macrophages in the lung, with upregulated expression of EndoMT markers in lung EdCs. Endothelial fate and function were investigated in vitro upon challenge with serum signals from SSc patients or released by activated macrophages. Sera of SSc patients with anti-Scl70 Abs, at higher risk of visceral organ fibrosis, induced EndoMT and jeopardized endothelial function. In conclusion, EdCs in SSc might be defective because of commitment to a mesenchymal fate, which is sustained by soluble signals in the patient's blood. Macrophages contribute to preserve the endothelial identity of precursor cells. Altered macrophage-dependent plasticity of EdCs could contribute to link vasculopathy with fibrosis.

The α4ß1/EMILIN1 interaction discloses a novel and unique integrin-ligand type of engagement.

EMILIN1, a homo-trimeric adhesive ECM glycoprotein, interacts with the α4ß1 integrin through its gC1q domain. Uniquely among the C1q family members, the EMILIN1 gC1q presents only nine-stranded ß-sandwich fold and the missing strand is substituted by a disordered 19-residue long segment spanning from Y927 to G945 at the apex of the gC1q domain. This unstructured loop exposes to the solvent the acidic residue E933, which plays a key role in the α4ß1 integrin mediated interaction. Here, we experimentally determined that the three E933 residues (one from each monomer) are all required for ligand binding. By docking the NMR structure of the gC1q to a virtual α4ß1 crystal structure based on the known structures of α4ß7 and α5ß1 integrins we built a model of α4ß1-gC1q complex where three E933 residues are smoothly forced to coordinate the Mg2+ ion at the ßI MIDAS site of the integrin. By bringing the three E933 close in space, the trimeric supramolecular organization of gC1q allows the formation of a proper 3D geometry and suggests a quaternary-structure-dependent mode of interaction. Furthermore, we experimentally identified R904 as a synergistic residue for cell adhesion. Accordingly, the model showed that this residue is able to form potential stabilizing intra-chain salt bridges with residues E928 and E930. This mode of interaction likely accounts for a more stable and durable α4ß1-gC1q interaction in comparison with the prototypic CS1 ligand. To our knowledge, this is the first report describing the simultaneous involvement of all the three acidic residues of a trimeric ligand in the formation of a dimeric complex with the integrin ßI domain.

Nitric Oxide Donor Molsidomine Positively Modulates Myogenic Differentiation of Embryonic Endothelial Progenitors.

Embryonic VE-Cadherin-expressing progenitors (eVE-Cad+), including hemogenic endothelium, have been shown to generate hematopoietic stem cells and a variety of other progenitors, including mesoangioblasts, or MABs. MABs are vessel-associated progenitors with multilineage mesodermal differentiation potential that can physiologically contribute to skeletal muscle development and regeneration, and have been used in an ex vivo cell therapy setting for the treatment of muscular dystrophy. There is currently a therapeutic need for molecules that could improve the efficacy of cell therapy protocols; one such good candidate is nitric oxide. Several studies in animal models of muscle dystrophy have demonstrated that nitric oxide donors provide several beneficial effects, including modulation of the activity of endogenous cell populations involved in muscle repair and the delay of muscle degeneration. Here we used a genetic lineage tracing approach to investigate whether the therapeutic effect of nitric oxide in muscle repair could derive from an improvement in the myogenic differentiation of eVE-Cad+ progenitors during embryogenesis. We show that early in vivo treatment with the nitric oxide donor molsidomine enhances eVE-Cad+ contribution to embryonic and fetal myogenesis, and that this effect could originate from a modulation of the properties of yolk sac hemogenic endothelium.

Vascular Remodelling and Mesenchymal Transition in Systemic Sclerosis.

Fibrosis of the skin and of internal organs, autoimmunity, and vascular inflammation are hallmarks of Systemic Sclerosis (SSc). The injury and activation of endothelial cells, with hyperplasia of the intima and eventual obliteration of the vascular lumen, are early features of SSc. Reduced capillary blood flow coupled with deficient angiogenesis leads to chronic hypoxia and tissue ischemia, enforcing a positive feed-forward loop sustaining vascular remodelling, further exacerbated by extracellular matrix accumulation due to fibrosis. Despite numerous developments and a growing number of controlled clinical trials no treatment has been shown so far to alter SSc natural history, outlining the need of further investigation in the molecular pathways involved in the pathogenesis of the disease. We review some processes potentially involved in SSc vasculopathy, with attention to the possible effect of sustained vascular inflammation on the plasticity of vascular cells. Specifically we focus on mesenchymal transition, a key phenomenon in the cardiac and vascular development as well as in the remodelling of injured vessels. Recent work supports the role of transforming growth factor-beta, Wnt, and Notch signaling in these processes. Importantly, endothelial-mesenchymal transition may be reversible, possibly offering novel cues for treatment.

Theranostic impact of NG2/CSPG4 proteoglycan in cancer.

NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be exploited in therapeutic interventions aimed at averting resistance to target therapy agents and at interfering with secondary lesion formation and/or tumour recurrence.

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

Fluorescence-based assays for in vitro analysis of cell adhesion and migration.

Cell adhesion and cell migration are two primary cellular phenomena for which in vitro approaches may be exploited to effectively dissect the individual events and underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also afford an efficient way of conducting larger basic and applied research screenings on the factors affecting these processes and are potentially exploitable in the context of routine diagnostic, prognostic, and predictive tests in the biological and medical fields. Therefore, there is a longstanding continuum in the interest in devising more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescence cell tagging, the design of instruments capable of detecting fluorescent signals with high sensitivity, and informatic tools allowing sophisticated elaboration of data generated through these instruments. In this report, we describe three representative fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automated for high-to-medium throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.

Proteoglycans in the control of tumor growth and metastasis formation.

Proteoglycans (PGs) as a whole, or when considering their GAG chains as single entities, are emerging as key regulators of tumor progression. Expectations on using them as putative prognostic markers and potential therapeutic targets are increasing coincidentally. Due to the multitude of biological roles that they may invest and the ample spectrum of cellular processes that they may control, we still need to learn better how they regulate phenomena such as intracellular signaling, proliferation, apoptosis, motility, and drug resistance. Depending on the type, their expression pattern, and the accessibility of their molecular ligands, PGs can either promote or inhibit tumorigenesis. The structural and functional diversity of PGs coupled with their ubiquitous abundance place them at the crossroads of many critical steps within the metastatic cascade. As this phenomenon is the pivotal factor for patient survivals, particular attention should be given to the understanding of how PGs govern metastasis formation.