Periostin expression and its supposed roles in benign and malignant thyroid nodules: an immunohistochemical study of 105 cases

Background Thyroid tumors are often difficult to histopathologically diagnose, particularly follicular adenoma (FA) and follicular carcinoma (FC). Papillary carcinoma (PAC) has several histological subtypes. Periostin (PON), which is a non-collagenous extracellular matrix molecule, has been implicated in tumor invasiveness. We herein aimed to elucidate the expression status and localization of PON in thyroid tumors. Method We collected 105 cases of thyroid nodules, which included cases of adenomatous goiter, FA, microcarcinoma (MIC), PAC, FC, poorly differentiated carcinoma (PDCa), and undifferentiated carcinoma (UCa), and immunohistochemically examined the PON expression patterns of these lesions. Results Stromal PON deposition was detected in PAC and MIC, particularly in the solid/sclerosing subtype, whereas FA and FC showed weak deposition on the fibrous capsule. However, the invasive and/or extracapsular regions of microinvasive FC showed quite strong PON expression. Except for it, we could not find any significant histopathological differences between FA and FC. There were no other significant histopathological differences between FA and FC. Although PDCa showed a similar PON expression pattern to PAC, UCa exhibited stromal PON deposition in its invasive portions and cytoplasmic expression in its carcinoma cells. Although there was only one case of UCa, it showed strong PON immunopositivity. PAC and MIC showed similar patterns of stromal PON deposition, particularly at the invasive front. Conclusions PON may play a role in the invasion of thyroid carcinomas, particularly PAC and UCa, whereas it may act as a barrier to the growth of tumor cells in FA and minimally invasive FC.

Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

Thrombospondin-4 (THBS4) is a non-structural extracellular matrix molecule associated with tissue regeneration and a variety of pathological processes characterized by increased cell proliferation and migration. However, the mechanisms of how THBS4 regulates cell behavior as well as the pathways contributing to its effects have remained largely unexplored. In the present study we investigated the role of THBS4 in skin regeneration both in vitro and in vivo. We found that THBS4 expression was upregulated in the dermal compartment of healing skin wounds in humans as well as in mice. Application of recombinant THBS4 protein promoted cutaneous wound healing in mice and selectively stimulated migration of primary fibroblasts as well as proliferation of keratinocytes in vitro. By using a combined proteotranscriptomic pathway analysis approach we discovered that β-catenin acted as a hub for THBS4-dependent cell signaling and likely plays a key role in promoting its downstream effects. Our results suggest that THBS4 is an important contributor to wound healing and its incorporation into novel wound healing therapies may be a promising strategy for treatment of cutaneous wounds.

Impact of Tenascin-C on Radiotherapy in a Novel Syngeneic Oral Squamous Cell Carcinoma Model With Spontaneous Dissemination to the Lymph Nodes

Radiotherapy, the most frequent treatment of oral squamous cell carcinomas (OSCC) besides surgery is employed to kill tumor cells but, radiotherapy may also promote tumor relapse where the immune-suppressive tumor microenvironment (TME) could be instrumental. We established a novel syngeneic grafting model from a carcinogen-induced tongue tumor, OSCC13, to address the impact of radiotherapy on OSCC. This model revealed similarities with human OSCC, recapitulating carcinogen-induced mutations found in smoking associated human tongue tumors, abundant tumor infiltrating leukocytes (TIL) and, spontaneous tumor cell dissemination to the local lymph nodes. Cultured OSCC13 cells and OSCC13-derived tongue tumors were sensitive to irradiation. At the chosen dose of 2 Gy mimicking treatment of human OSCC patients not all tumor cells were killed allowing to investigate effects on the TME. By investigating expression of the extracellular matrix molecule tenascin-C (TNC), an indicator of an immune suppressive TME, we observed high local TNC expression and TIL infiltration in the irradiated tumors. In a TNC knockout host the TME appeared less immune suppressive with a tendency towards more tumor regression than in WT conditions. Altogether, our novel syngeneic tongue OSCC grafting model, sharing important features with the human OSCC disease could be relevant for future anti-cancer targeting of OSCC by radiotherapy and other therapeutic approaches.

Neural crest cell-derived pericytes act as pro-angiogenic cells in human neocortex development and gliomas

Central nervous system diseases involving the parenchymal microvessels are frequently associated with a ‘microvasculopathy’, which includes different levels of neurovascular unit (NVU) dysfunction, including blood–brain barrier alterations. To contribute to the understanding of NVU responses to pathological noxae, we have focused on one of its cellular components, the microvascular pericytes, highlighting unique features of brain pericytes with the aid of the analyses carried out during vascularization of human developing neocortex and in human gliomas. Thanks to their position, centred within the endothelial/glial partition of the vessel basal lamina and therefore inserted between endothelial cells and the perivascular and vessel-associated components (astrocytes, oligodendrocyte precursor cells (OPCs)/NG2-glia, microglia, macrophages, nerve terminals), pericytes fulfil a central role within the microvessel NVU. Indeed, at this critical site, pericytes have a number of direct and extracellular matrix molecule- and soluble factor-mediated functions, displaying marked phenotypical and functional heterogeneity and carrying out multitasking services. This pericytes heterogeneity is primarily linked to their position in specific tissue and organ microenvironments and, most importantly, to their ontogeny. During ontogenesis, pericyte subtypes belong to two main embryonic germ layers, mesoderm and (neuro)ectoderm, and are therefore expected to be found in organs ontogenetically different, nonetheless, pericytes of different origin may converge and colonize neighbouring areas of the same organ/apparatus. Here, we provide a brief overview of the unusual roles played by forebrain pericytes in the processes of angiogenesis and barriergenesis by virtue of their origin from midbrain neural crest stem cells. A better knowledge of the ontogenetic subpopulations may support the understanding of specific interactions and mechanisms involved in pericyte function/dysfunction, including normal and pathological angiogenesis, thereby offering an alternative perspective on cell subtype-specific therapeutic approaches.

Periostin Expression and Its Roles in Benign and Malignant Thyroid Nodules: an Immunohistochemical Study of 105 Cases

Background. Thyroid tumors are often difficult to histopathologically diagnose, especially follicular adenoma (FA) and follicular carcinoma (FC). Papillary carcinoma (PAC) has several histological subtypes. Periostin (PON), which is a non-collagenous extracellular matrix molecule, is related to tumor invasiveness. We aimed to elucidate the role of PON in thyroid tumors.Method. We collected 105 cases of thyroid nodules, which included cases of adenomatous goiter, FA, microcarcinoma (MIC), PAC, FC, poorly differentiated carcinoma (PDCa), and undifferentiated carcinoma (UCa), and immunohistochemically examined the PON expression patterns of these lesions. Results. PAC and MIC exhibited stromal PON deposition, especially in the solid/sclerosing subtype, whereas FA and FC showed weak deposition on the fibrous capsule. However, the invasive and/or extracapsular regions of microinvasive FC demonstrated quite strong PON expression.Except for it, we could not find any significantly histopathological differences between FA and FC. Although PDCa showed a similar PON expression pattern to PAC, UCa exhibited stromal PON deposition in its invasive portions and cytoplasmic expression in its carcinoma cells. Although there was only one case of UCa, it demonstrated strong PON immunopositivity. PAC and MIC showed similar patterns of stromal PON deposition, especially at the invasive front.Conclusions. PON plays a role in the invasion of thyroid carcinomas, especially PAC and UCa, whereas it acts as a barrier against the growth of tumor cells in FA and minimally invasive FC.

Perlecan Facilitates Neuronal Nitric Oxide Synthase Delocalization in Denervation-Induced Muscle Atrophy

Perlecan is an extracellular matrix molecule anchored to the sarcolemma by a dystrophin–glycoprotein complex. Perlecan-deficient mice are tolerant to muscle atrophy, suggesting that perlecan negatively regulates mechanical stress-dependent skeletal muscle mass. Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol triggers protein degradation, thereby initiating skeletal muscle atrophy. We hypothesized that perlecan regulates nNOS delocalization and activates protein degradation during this process. To determine the role of perlecan in nNOS-mediated mechanotransduction, we used sciatic nerve transection as a denervation model of gastrocnemius muscles. Gastrocnemius muscle atrophy was significantly lower in perinatal lethality-rescued perlecan-knockout (Hspg2−/−-Tg) mice than controls (WT-Tg) on days 4 and 14 following surgery. Immunofluorescence microscopy showed that cell membrane nNOS expression was reduced by denervation in WT-Tg mice, with marginal effects in Hspg2−/−-Tg mice. Moreover, levels of atrophy-related proteins—i.e., FoxO1a, FoxO3a, atrogin-1, and Lys48-polyubiquitinated proteins—increased in the denervated muscles of WT-Tg mice but not in Hspg2−/−-Tg mice. These findings suggest that during denervation, perlecan promotes nNOS delocalization from the membrane and stimulates protein degradation and muscle atrophy by activating FoxO signaling and the ubiquitin–proteasome system.

The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus’s life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect.

Loss of the extracellular matrix molecule tenascin-C leads to absence of reactive gliosis and promotes anti-inflammatory cytokine expression in an autoimmune glaucoma mouse model

Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc).Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. 10 weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells and the expression of different cytokines in retina and optic nerve tissue in all four groups.IOP and retinal function was comparable in all groups. Although less severe in KO ONA, WT and KO mice displayed a significant loss of RGCs after immunization. Compared to KO ONA, a significant reduction of βIII-tubulin stained axons and oligodendrocyte markers was noted in the optic nerve of WT ONA. In retinal and optic nerve slices, we found an enhanced GFAP+ staining area of astrocytes in immunized WT. In retinal flat-mounts, a significantly higher number of Iba1+ microglia was found in WT ONA, while a lower number of Iba1+ cells was observed in KO ONA. Furthermore, an increased expression of the glial markers Gfap, Iba1, Nos2 and Cd68 was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA post immunization. In addition, pro-inflammatory Tnfa expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory Tgfb expression was measured in KO ONA animals.Collectively, this study revealed that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model offers for the first time the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling.

Quantification of the Extracellular Matrix Molecule Thrombospondin 1 and Its Pericellular Association in the Brain Using a Semiautomated Computerized Approach

The structure and functions of the extracellular matrix (ECM), its spatial distribution and pericellular association of ECM molecules remain poorly understood. Colocalization of ECM molecules with cell phenotypes through immunohistochemistry can provide crucial insights into their juxtacrine signaling role as well as their structural relevance to tissue architecture. As manual quantification of images introduces intra- and inter-user bias and is cumbersome for high-throughput approaches, we implemented an automated high-throughput method to quantify the spatial distribution and cellular association of one ECM molecule, thrombospondin 1 (TSP1) with two major cell phenotypes, neurons, and astrocytes. The distribution of TSP1 was homogeneous throughout the striatum and cortex along the anterior–posterior axis. TSP1 occupied 8.85% of the striatum and 7.40% in the cortex. TSP1 also associated with 94.58% and 88.45% of neurons in the striatum and cortex. The association with astrocytes was significantly lower at 47.55% and 28.09%. These findings highlight the key role that TSP1 plays in neuron physiology in a healthy brain, but also highlights key regional difference in astrocytes secreting ECM molecules. The semiautomated approach implemented here will improve the throughput and reliability of measuring the distribution and cellular colocalization of ECM molecules.