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.

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.

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.

STAT3, tumor microenvironment, and microvessel density in diffuse large B cell lymphomas.

Constitutively activated STAT3 is correlated with more advanced clinical stage and overall poor survival of diffuse large B-cell lymphoma (DLBCL). The aim of this study was to evaluate STAT3 and Ki67 tumor cell expression, inflammatory cell infiltration, microvascular density in DLBCL bioptic specimens. RNA-scope showed that activated B cell (ABC) tissue samples contained a significant higher number of STAT3+ cells as compared to germinal center B (GCB) tissue samples. Immunohistochemical analysis showed a significant increased levels of CD3, CD8, CD68, CD163, CD34, and Ki67 positive cells in ABC patients. A positive correlation between STAT3 and CD3, CD8, CD68, and CD163 was evidenced in ABC group. In ABC group, we found also a positive correlation between CD8 and CD34 and a positive correlation between Ki67 and, CD68, and CD163. These data indicate that in ABC-as compared to GCB-DLBCL, a higher STAT3 expression is associated with a higher CD163+ TAM and CD8+ cell infiltration which induces a strong angiogenic response.

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.

Dp71 Expression in Human Glioblastoma.

BACKGROUND: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). METHODS: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. RESULTS: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. CONCLUSION: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.

PTX3 Modulates Neovascularization and Immune Inflammatory Infiltrate in a Murine Model of Fibrosarcoma.

Fibrosarcoma is an aggressive subtype of soft tissue sarcoma categorized in infantile/congenital-type and adult-type. Fibrosarcoma cells and its surrounding immune inflammatory infiltrates overexpress or induce the expression of fibroblast growth factor-2 (FGF-2) that have a crucial role in tumor progression and angiogenesis. The inflammation-associated long pentraxin 3 (PTX3) was found to reduce FGF-2-mediated angiogenesis, but its role on fibrosarcoma immune inflammatory infiltrate is still unknown. In this study, we have evaluated the PTX3 activity on immune infiltrating mast cells, macrophages and T-lymphocytes by immunohistochemistry on murine MC-TGS17-51 fibrosarcoma cells and on transgenic TgN(Tie2-hPTX3) mouse. In these fibrosarcoma models we found a reduced neovascularization and a significant decrease of inflammatory infiltrate. Indeed, we show that PTX3 reduces the level of complement 3 (C3) deposition reducing fibrosarcoma progression. In conclusion, we hypothesize that targeting fibrosarcoma microenvironment by FGF/FGFR inhibitors may improve treatment outcome.

Limitations of Anti-Angiogenic Treatment of Tumors.

Clinical trials using anti-vascular endothelial growth factor /(VEGF) molecules induce a modest improvement in overall survival, measurable in weeks to just a few months, and tumors respond differently to these agents. In this review article, we have exposed some tumor characteristics and processes that may impair the effectiveness of anti-angiogenic approaches, including genotypic changes on endothelial cells, the vascular normalization phenomenon, and the vasculogenic mimicry. The usage of anti-angiogenic molecules leads to hypoxic tumor microenvironment which enhances tumor invasiveness. The role of tumor-infiltrating cells, including tumor associated macrophages and fibroblasts (TAMs and TAFs) in the therapeutic response to anti-angiogenic settings was also highlighted. Finally, among the new therapeutic approaches to target tumor vasculature, anti-PD-1 or anti-PD-L1 therapy sensitizing and prolonging the efficacy of anti-angiogenic therapy, have been discussed.

Angiogenesis in Pancreatic Cancer: Pre-Clinical and Clinical Studies.

Angiogenesis is a crucial event in tumor development and progression, occurring by different mechanisms and it is driven by pro- and anti-angiogenic molecules. Pancreatic cancer vascularization is characterized by a high microvascular density, impaired microvessel integrity and poor perfused vessels with heterogeneous distribution. In this review article, after a brief introduction on pancreatic cancer classification and on angiogenesis mechanisms involved in its progression, the pre-clinical and clinical trials conducted in pancreatic cancer treatment using anti-angiogenic inhibitors will be described. Finally, we will discuss the anti-angiogenic therapy paradox between the advantage to abolish vessel supply to block tumor growth and the disadvantage due to reduction of drug delivery at the same time. The purpose is to identify new anti-angiogenic molecules that may enhance treatment regimen.

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.

α-Methyl-prednisolone normalizes the PKC mediated brain angiogenesis in dystrophic mdx mice.

A fraction of patients affected by Duchenne Muscular Dystrophy (DMD) shows mental disability as a consequence of neuronal and metabolic alteration. In this study, we evaluated the effect of α-methyl-prednisolone (PDN) on the expression of the angiogenic marker HIF1α, VEGFA and VEGFR-2 (FLK1) in correlation with PKC expression in the brain of mdx mouse, an experimental model of DMD. We demonstrated that HIF1α, VEGFA and FLK1 are overexpressed in the brain of dystrophic mdx mice in parallel with an increase of PKC expression and reduction of the tight junctions Occludin leading to altered angiogenesis. Moreover, we demonstrated that PDN treatment induces a significant reduction in the HIF1α, VEGF, FLK1, and PKC mRNA and proteins levels and restores Occludin expression reducing its phosphorylation pattern. Our results suggest a new mechanism of action of PDN that through PKC suppression normalizes the angiogenesis in dystrophic mdx brains.

Microvascular density, macrophages, and mast cells in human clear cell renal carcinoma with and without bevacizumab treatment.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) represents a highly vascularized aggressive kidney cancer. Due to ccRCC chemotherapy resistance, antiangiogenesis is one of the most innovative targeted therapies for this tumor. The tumor microenvironment exerts important roles in tumor growth, angiogenesis, and metastatic escape. MATERIALS AND METHODS: In this study, we investigated the composition of tumor cell microenvironment including mast cells, macrophages, and microvascular density in ccRCC tumor tissues collected from patients who underwent nephrectomy treated or not with bevacizumab as neoadjuvant therapy before surgery. RESULTS: The results of this study indicate that bevacizumab-treated ccRCC samples present reduced microvascular density as well as a lower number of CD68-positive macrophages and tryptase-positive mast cells in comparison with the untreated patients. CONCLUSIONS: It follows that the antiangiogenic activity of bevacizumab may be due to a direct effect on angiogenic cytokines released by tumor cells and an indirect effect on the release of pro-angiogenic factors by inflammatory stromal cells.

DP71 and SERCA2 alteration in human neurons of a Duchenne muscular dystrophy patient.

Cognitive deficit has been identified in one third of patients affected by Duchenne Muscular Dystrophy, primarily attributed to loss of the short Dp71 dystrophin, the major brain dystrophin isoform. In this study, we investigated for the first time the Dp71 and Dp71-associated proteins cellular localization and expression in human neurons obtained by differentiation from induced pluripotent stem cell line of a patient affected by cognitive impairment. We found structural and molecular alterations in both pluripotent stem cell and derived neurons, reduced Dp71 expression, and a Ca2+ cytoplasmic overload in neurons coupled with increased expression of the SERCA2 pump in the dystrophic neurons. These results suggest that the reduction of Dp71 protein in the Duchenne muscular dystrophy neurons leads to alterations in SERCA2 and to elevated cytosolic Ca2+ concentration with consequent potential disruption of the dystrophin proteins and Dp71-associated proteins.

STAT-3 RNAscope Determination in Human Diffuse Large B-Cell Lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription with many important functions, including regulation of cell proliferation, differentiation, survival, angiogenesis, and immune response. MATERIALS AND METHODS: In this study, we have compared by means of RNAscope technology STAT3 RNA expression in human DLBCL in a selected group of activated B-cell-like DLBCL (ABC-DLBCL) patients with another group of germinal center B-cell-like DLBCL (GBC-DLBCL) patients. RESULTS: The results have shown that ABC DLBCL tissue samples contained a significantly higher number of STAT3-positive cells as compared with GCB tissue samples. Moreover, by means of confocal immunofluorescence analysis, we have observed that tumor vessels in ABC samples appeared lined by endothelial cells expressing both FVIII and STAT3 signals, while in GCB samples, only few vessels coexpressed FVIII and STAT3. CONCLUSIONS: These data confirm other reports showing that STAT3 is highly expressed and activated in ABC-DLBCL and our previously published data demonstrating that, in primary central nervous system lymphoma, tumor vessels appeared lined by endothelial cells expressing both FVIII and STAT3.

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.

Mast cells and primary systemic vasculitides.

Vasculitides are characterized by inflammation and necrosis of blood vessels leading to vessel occlusion and ischemic damages of tissues. Among the inflammatory cells involved in vasculitides, neutrophils, T cells, and macrophages have been identified as the predominant cell type. This review article is focused on the role of mast cells in these chronic inflammatory processes. Mast cells are characterized by their complex plasticity. Increasing evidences document that mast cells exert both pro- and anti-inflammatory functions depending on the cell types and the microenvironment they reside in. In this context, mast cell mediators able to modulate progression of vasculitides at different levels and the anatomic localization of mast cells in different vasculitides will be described. Finally, therapeutic approach including inhibition of recruitment of mast cells to the inflammatory infiltrate and blockade of their proinflammatory effects and proangiogenic functions as potential new targets for the treatment of these diseases will be discussed.

Bcl6/p53 expression, macrophages/mast cells infiltration and microvascular density in invasive breast carcinoma.

To better understand the breast cancer progression and therapeutic resistance is crucial deepen the molecular mechanisms related to regulation of cells behavior in the tumor microenvironment. Inappropriate expression or activation of transcription factors in tumor breast microenvironment can lead to the malignant behavior of breast cancer cells. Bcl6 is a transcriptional factor that may play a role in the pathogenesis of breast cancer. Moreover, cells surrounding tumor cells, including macrophages and mast cells play an important role during tumor progression enhancing angiogenesis. We have demonstrated: 1) An increase of the BCL6 translocation and Bcl6 positive cells in G3 degree of disease; 2) A reduction of the expression of p53 in G3 breast cancer samples as compared to G1/G2 specimens; 3) Macrophages CD68+ and CD163+ in interstitial and periglandular position, increase in G3 specimens as compared to G1/G2 and control samples; 4) Tryptase-positive mast cells in periglandular position are more numerous in G3 tumor specimens as compared to G1/G2 and control samples. Overall, these data confirm the important role played by epigenetic events, including BCL6 translocation, p53 expression, and microenvironment components, including macrophage and mast cell infiltration and microvascular density involved in the regulation of breast cancer progression.

VEGFA and VEGFR2 RNAscope determination in gastric cancer.

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.

Rhu-Epo down-regulates pro-tumorigenic activity of cancer-associated fibroblasts in multiple myeloma.

We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.

Reduced myofilament component in primary Sjögren's syndrome salivary gland myoepithelial cells.

Primary Sjögren's syndrome (pSS) is a solitary poorly understood autoimmune inflammatory disease by involvement of the salivary and lacrimal glands resulting in dry mouth and dry eyes. Myoepithelial cells (MECs) are cells knowing for its hybrid epithelial and mesenchymal phenotype that are important components of the salivary gland (SGs) structure aiding the expulsion of saliva from acinar lobules. In this study we investigate possible alteration in the myofilament component of MECs in SGs specimens obtained from pSS patients in comparison with healthy subjects, to evaluate MECs hypothetical involvement in the pathogenesis of pSS. The expression of alpha-smooth muscle actin (α-SMA) and p63, as MECs markers, was evaluated in bioptic specimens from pSS and healthy labial SGs through immunohistochemistry and immunofluorescence analyses; the distribution of MECs markers was quantified using Aperio ScanScope and ImageScope software to provide quantitative assessments of staining levels. Our observations demonstrated that p63 nuclear labeling in pSS MECs is preserved whereas α-SMA cytoplasmic staining is strongly and significantly reduced when compared with healthy SGs; the digital images analysis quantification of the expression of labeled α-SMA and p63 protein in the healthy and pSS MECs salivary tissues, led to results suggesting a loss of mechanical support for acini and ducts in pSS, correlated, probably, with the reduction of salivary flow that features one important aspect of pSS disease.