MUC1 Expression Affects the Immunoflogosis in Renal Cell Carcinoma Microenvironment through Complement System Activation and Immune Infiltrate Modulation.

Mucin1 (MUC1), a glycoprotein associated with an aggressive cancer phenotype and chemoresistance, is aberrantly overexpressed in a subset of clear cell renal cell carcinoma (ccRCC). Recent studies suggest that MUC1 plays a role in modulating cancer cell metabolism, but its role in regulating immunoflogosis in the tumor microenvironment remains poorly understood. In a previous study, we showed that pentraxin-3 (PTX3) can affect the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system (C1q) and releasing proangiogenic factors (C3a, C5a). In this scenario, we evaluated the PTX3 expression and analyzed the potential role of complement system activation on tumor site and immune microenvironment modulation, stratifying samples in tumors with high (MUC1H) versus tumors with low MUC1 expression (MUC1L). We found that PTX3 tissue expression was significantly higher in MUC1H ccRCC. In addition, C1q deposition and the expressions of CD59, C3aR, and C5aR were extensively present in MUC1H ccRCC tissue samples and colocalized with PTX3. Finally, MUC1 expression was associated with an increased number of infiltrating mast cells, M2-macrophage, and IDO1+ cells, and a reduced number of CD8+ T cells. Taken together, our results suggest that expression of MUC1 can modulate the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system and regulating the immune infiltrate, promoting an immune-silent microenvironment.

The role of vascular niche and endothelial cells in organogenesis and regeneration.

The term vascular niche indicate the physical and biochemical microenvironment around blood vessel where endothelial cells, pericytes, and smooth muscle cells organize themselves to form blood vessels and release molecules involved in the recruitment of hematopoietic stem cells, endothelial progenitor cells and mesenchymal stem cells. The vascular niche creates a permissive environment that enables different cell types to realize their developmental or regenerative programs. In this context, the proximity between the endothelium and the new-forming cellular components of organs suggests an essential role of endothelial cells in the organs maturation. Dynamic interactions between specific organ endothelial cells and different cellular conponents are crucial for different organ morphogenesis and function. Conversely, organs provide cues shaping vascular network structure.

Chorioallantoic membrane vascularization. A meta-analysis.

The CAM is a widely used experimental assay to study angiogenesis, wound healing, tumor growth and metastatic process. In this study, we have analyzed and compared the existent literature data concerning the growth of the CAM. Moreover, we have analyzed the data concerning the development of the vascular system and the expression of the most important pro-angiogenic and anti-angiogenic factors. The availability of these data and their comparative evaluation allow to better analyze the experimental data concerning the testing of different pro-angiogenic and anti-angiogenic molecules, as well as biomaterials in the CAM assay. Moreover, the dynamic of the angiogenic response to different tumor cell lines and or tumor bioptic specimens, may be also better evaluated and estimated.

Nutraceuticals and their role in tumor angiogenesis.

Angiogenesis plays a pivotal role in cancer initiation, maintenance, and progression. Diet may inhibit, retard or reverse these processes affecting angiogenesis (angioprevention). Nutraceuticals, such as omega-3 fatty acids, amino acids, proteins, vitamins, minerals, fibers, and phenolic compounds, improve health benefits as they are a source of bioactive compounds that, among other effects, can regulate angiogenesis. The literature concerning the pro-angiogenic and/or anti-angiogenic nutraceuticals and the possible activated pathways in cancer and other non-neoplastic diseases by in vivo and in vitro experiments are reviewed.

Oxytocin regulates body composition.

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre+:Oxtrfl/fl mice recapitulate the low-bone mass phenotype of Oxtr+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre+:Oxtrfl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre+:Oxtrfl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre+:Oxtrfl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt-/- and Oxtr-/- mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

Epigenetic control of tumor angiogenesis.

The term "epigenetic" is used to refer to heritable alterations in chromatin that are not due to changes in DNA sequence. Different growth factors and vascular genes mediate the angiogenic process, which is regulated by epigenetic states of genes. The aim of this article is to analyze the role of epigenetic mechanisms in the control and regulation of tumor angiogenetic processes. The reversibility of epigenetic events in contrast to genetic aberrations makes them potentially suitable for therapeutic intervention. In this context, DNA methyltransferase (DNMT) and HDAC inhibitors indirectly-via the tumor cells-exhibit angiostatic effects in vivo, and inhibition of miRNAs can contribute to the development of novel anti-angiogenesis therapies.

Surface markers: An identity card of endothelial cells.

All endothelial cells have the common characteristic that they line the vessels of the blood circulatory system. However, endothelial cells display a large degree of heterogeneity in the function of their location in the vascular tree. In this article, we have summarized the expression patterns of a number of well-accepted endothelial surface markers present in normal microvascular endothelial cells, arterial and venous endothelial cells, lymphatic endothelial cells, tumor endothelial cells, and endothelial precursor cells.

Combined Replenishment of miR-34a and let-7b by Targeted Nanoparticles Inhibits Tumor Growth in Neuroblastoma Preclinical Models.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.

RNAscope dual ISH-IHC technology to study angiogenesis in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) are the most common types of Non-Hodgkin's lymphomas and are highly heterogeneous in terms of phenotype and treatment response. The natural course of DLBCLs tumor progression is featured by a flow of events leading to the enhancement of proliferative and invasive capabilities and, therefore, towards the establishment of a more aggressive phenotype. Angiogenesis is a constant hallmark of DLBCLs progression, has prognostic potential and promote DLBCLs dissemination. The study of DLBCLs angiogenesis mechanisms, and the tumor endothelium characterization, will allow us to identify new prognostic/predictive biomarkers to proper patient selection to antiangiogenic treatment. In our previous work, by RNAscope technology, we have demonstrated that Janus kinase (Jak) and signal transducer activator of transcription pathway (STAT) is one of the proangiogenic pathways activated in DLBCLs and it drives neoangiogenesis occurred by vasculogenesis mechanism. Here, we describe a detailed protocol to perform RNAscope technology alone and in combination with immunohistochemistry (called dual RNAscope ISH-IHC) in DLBCLs formalin-fixed, paraffin-embedded sections. We propose dual ISH-IHC as an extremely powerful method to study angiogenesis in DLBCLs, because it allows one to answer important biological questions that are difficult to address using other single methods.

The use of the chick embryo CAM assay in the study of angiogenic activiy of biomaterials.

The chick embryo chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane, which carries out several functions during embryonic development, including exchange of respiratory gases, calcium transport from the eggshell, acid-base homeostasis in the embryo, and ion and water reabsorption from the allantoic fluid. Due to its easy accessibility, affordability and given that it constitutes an immunodeficient environment, CAM has been used as an experimental model for >50 years and in particular it has been broadly used to study angiogenesis and anti-angiogenesis. This review article describes the use of the CAM assay as a valuable assay to test angiogenic activity of biomaterials in vivo before they are further investigated in animal models. In this context, the use of CAM has become an integral part of the biocompatibility testing process for developing potential biomaterials.

Morphometric analysis of the branching of the vascular tree in the chick embryo area vasculosa.

The chick embryo includes the area vasculosa is subdivided into 2 concentric zones, the inner transparent area pellucida vasculosa and the surrounding less transparent area opaca vasculosa, peripherally limited by the sinus terminalis. In this study, we have analyzed by a modern morphometric approach the total length of the vascular network, the number of vascular branches, of the branching points density, the modality of vessel ramification, and spatial arrangement of the vascular network in four consecutive stages of development of the area vasculosa. The results have shown that there is a significant 15% increase in the total length of the vascular network associated with a progressive increase of the number of vascular branches and of the branching points density. Moreover, the results indicated that vascular spatial disorder significantly decreased during development in area vasculosa, suggesting a more uniform occupancy of the tissue by the vascular pattern. Finally, a more regular pattern of branching was observed, as indicated by the significant decrease of topological disorder of the vascular tree.

Mast cells and angiogenesis in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease, characterized by multiple demyelination of axons in both white and gray matter in the Central Nervous System (CNS). There is increasing evidence to support the notion that angiogenesis and chronic inflammation are mutually related. Different immune cells, including monocytes-macrophages, lymphocytes, neutrophils, mast cells (MCs) and dendritic cells are able to secrete an array of angiogenic cytokines, which promote growth, migration, and activation of endothelial cells. MCs play various roles in MS pathogenesis, influencing the innate immune response in peripheral tissues and in CNS. The aim of this review article is to discuss the role of MCs in MS pathogenesis with particular reference to the involvement of these inflammatory cells in the angiogenic processes occurring during MS.

The morphological basis of the development of the chick embryo immune system.

The chick immune system is a fundamental model in basic immunology. In birds, the bone marrow derived pluripotent stem cells after entering the circulation, migrate to bursa of Fabricius to benefit from a microenvironment which supports the differentiation and maturation of B lymphocytes by the help of its resident cells and tissues. Delivering sufficient functional B cells is required to maintain their peripheral population and normal peripheral humoral responses. Additionally, bursa acts as an active site for the generation of antibody diversity through gene conversion. Being consisted of 98% B lymphocytes, the organ is occupied by other cell types including T cells, macrophages, eosinophils and mast cells. Thymus, which is an epithelial organ is the main site of T cell development where positive and negative selections contribute to the development of functional and not autoreactive T cell repertoire. Bursectomy and thymectomy are surgical exercises through which the involvement of cells of specific immunity including B cells and T cells can be determined.

Erythropoietin in tumor angiogenesis.

Erythropoietin (EPO) is a moonlighting protein since is ability to work as hormone, cytokine and growth factor. Its cardinal function is to regulate erythropoiesis in the bone marrow. However, EPO with his receptor EPOR are expressed also in non-hematopoietic tissues such as endothelium where they exert a protective function. Moreover, it is known EPO-EPOR pathway contribute to neovascularization in the angiogenic switch of tumor, but the mechanism is not completely established. In this article, after a brief introduction on tumor angiogenesis and description of classical and non-classical pro-angiogenic factors, we review the role of EPO in tumor angiogenesis highlighting the different mechanisms activated by it to promote tumor growth and progression. Finally, we analyze the controversy between the beneficial and the harmful effects of EPO. We suppose that the accurate characterization of EPO variants and their downstream pathways will allow to develop specific inhibition strategies to block only EPOR expressed by tumor cells without inducing signalling in hematopoietic cells to avoid side effects.

Vascular Wall as Source of Stem Cells Able to Differentiate into Endothelial Cells.

The traditional view of the vascular biology is changed by the discovery of vascular progenitor cells in bone marrow or peripheral blood Further complexity is due to the findings that the vessel walls harbor progenitor and stem cells, called vascular wall-resident vascular stem cells (VW-VSCs), able to differentiate to mature vascular wall cells. These immature stem/progenitor cell populations and multipotent mesenchymal lineage participate in postnatal neovascularization and vascular wall remodeling. Further studies are necessary to deepen the knowledge on characterization and biology of VW-VSCs, in particular of endothelial progenitor cells (EPCs) in order to improve their use in clinical settings for regenerative approaches.

Inflammatory cells infiltrate and angiogenesis in locally advanced and metastatic cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is the second most common subtype of primary hepatobiliary cancer and one of the most aggressive characterized by an extremely poor prognosis with limited treatment options. Inflammatory cells in tumour microenvironment support tumour growth in term of progression, angiogenesis and metastatic capacity. A link between inflammation and biliary carcinogenesis has been previously observed but the mechanisms involved remain to be determined. METHODS: We investigated the microvascular density (MVD) and inflammatory cells in tissue samples from 40 patients with CCA with locally advanced CCA and metastatic CCA by means of immunohistochemical analysis of macrophages, mast cells, B and T lymphocytes and we correlated inflammatory infiltrate with MVD. RESULTS: We observed significant decrease in the levels of CD31 positive vessels, and CD8, CD4, CD68 and tryptase-positive cells in metastatic lesions as compared to the localized ones. A negative correlation between CD31 and CD8 and CD31 and CD4 in localized CCA samples was found as assessed by Spearman correlation analysis. CONCLUSIONS: In locally advanced CCA patients, there is a significant increase of immune cell infiltrate constituted by CD8+ and CD4+ lymphocytes, macrophages and mast cells as compared to the metastatic ones. This alteration in the tumour microenvironment infiltrate is related to a significant increased MVD in localized CCA lesions compared with the metastatic ones. Moreover, we observed a negative correlation between MVD and CD8+ , CD4+ cells in localized CCA patients.

Vascular density and inflammatory infiltrate in primary oral squamous cell carcinoma and after allogeneic hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) recipients have been reported to have an increased risk of chronic graft versus host disease (cGVHD) and hematological and solid cancers. Oral manifestations are the first signs of cGVHD observed in the majority of patients, and oropharyngeal cancer is the most frequent secondary malignancy occurred after HSCT. In this study, we have evaluated the inflammatory infiltrate cell content and correlated with the vascular density in patients affected by primary oral squamous cell carcinoma (OSCC) from previous healthy controls and OSCC after cGVHD. Results have demonstrated that patients with OSCC after GVHD show a more consistent inflammatory infiltrate as compared with the OSCC ones. In detail, the inflammatory background composed of CD3-positive T cells, tryptase-positive mast cells, CD31-positive endothelial cells, and CD68-positive macrophages may be more pronounced in the setting of GVHD + OSCC than in the control group. By contrast, CD20-positive B cells and CD1a-positive dendritic cells were more abundant in the latter population. Finally, a positive correlation was found as between vascular density and inflammatory cell infiltration in both GVHD + OSCC and OSCC groups. Overall, these results confirm the role played by immune cells in enhancing tumor progression and angiogenesis and suggest a potential therapeutic strategy involving inhibition of recruitment of immune cells to the tumor microenvironment and blockade of pro-tumoral effects and pro-angiogenic functions.

Giulio Gabbiani and the discovery of myofibroblasts.

Myofibroblasts, specialized fibroblasts expressing the protein alpha-smooth muscle actin, are instrumental in wound contraction during normal wound healing. Tissue shortening is then stabilized by the synthesis of extracellular matrix, collagen in particular. Alpha-smooth muscle actin within myofibroblasts becomes organized in filamentous bundles, called stress fibers, that allow the retractile movement producing wound contraction. During hypertrophic scarring, skin deformations depend on the inappropriate action of these stress fibers that for unknown reasons persist even after the epithelialization of the wound. This historical review article is dedicated to the reconstruction of the discovery of this cell by the Italian scientist Giulio Gabbiani.

Cross talk between natural killer cells and mast cells in tumor angiogenesis.

Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. Under pathological conditions and during inflammation, NK cells extravasate into the lymph nodes and accumulate at inflammatory or tumor sites. The activation of NK cells depends on an intricate balance between activating and inhibitory signals that determines if a target will be susceptible to NK-mediated lysis. Many experimental evidences indicate that NK cells are also involved in several immunoregulatory processes and have the ability to modulate the adaptive immune responses. Many other important aspects about NK cell biology are emerging in these last years. The aim of this review is to elucidate the role of NK cells in tumor angiogenesis and their interaction with mast cells. In fact, it has been observed that NK cells produce pro-angiogenic factors and participate alone or in cooperation with mast cells to the regulation of angiogenesis in both physiological and pathological conditions including tumors.

Functions of vasopressin and oxytocin in bone mass regulation.

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr(-/-) mice have osteopenia, and Avpr1α(-/-) mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr(-/-):Avpr1α(-/-) double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α(-/-) cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.