Beatrice Nico

Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma

SummaryImmunohistochemical, cytochemical and ultrastructural data showing vivid angiogenesis and numerous mast cells (MCs) in the bone marrow of 24 patients with active multiple myeloma (MM) compared with 34 patients with non-active MM and 22 patients with monoclonal gammopathy of undetermined significance (MGUS) led us to hypothesize that angiogenesis parallels progression of MM, and that MCs participate in its induction via angiogenic factors in their secretory granules.

Bortezomib Mediates Antiangiogenesis in Multiple Myeloma via Direct and Indirect Effects on Endothelial Cells

Bone marrow angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma. Recent studies have shown that proteasome inhibitor bortezomib (Velcade, formerly PS-341) can overcome conventional drug resistance in vitro and in vivo; however, its antiangiogenic activity in the bone marrow milieu has not yet been defined. In the present study, we examined the effects of bortezomib on the angiogenic phenotype of multiple myeloma patient-derived endothelial cells (MMEC). At clinically achievable concentrations, bortezomib inhibited the proliferation of MMECs and human umbilical vein endothelial cells in a dose-dependent and time-dependent manner. In functional assays of angiogenesis, including chemotaxis, adhesion to fibronectin, capillary formation on Matrigel, and chick embryo chorioallantoic membrane assay, bortezomib induced a dose-dependent inhibition of angiogenesis. Importantly, binding of MM.1S cells to MMECs triggered multiple myeloma cell proliferation, which was also abrogated by bortezomib in a dose-dependent fashion. Bortezomib triggered a dose-dependent inhibition of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) secretion by the MMECs, and reverse transcriptase-PCR confirmed drug-related down-regulation of VEGF, IL-6, insulin-like growth factor-I, Angiopoietin 1 (Ang1), and Ang2 transcription. These data, therefore, delineate the mechanisms of the antiangiogenic effects of bortezomib on multiple myeloma cells in the bone marrow milieu.

Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo.

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti‐angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAMs capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor‐2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti‐angiogenesis. Anat Rec 264:317–324, 2001.

The role of pericytes in angiogenesis

Pericytes are branched cells embedded within the basement membrane of capillaries and post-capillary venules. They provide an incomplete investment to endothelial cells, thus reinforcing vascular structure and regulating microvascular blood flow. Pericytes exert an important role on endothelial cell proliferation, migration and stabilization. Endothelial cells, in turn, stimulate expansion and activation of the pericyte precursor cell population. The balance between the number of endothelial cells and pericytes is highly controlled by a series of signaling pathway mechanisms operating in an autocrine and/or paracrine manner. In this review, we will first examine the molecular aspects of the pericyte activating factors secreted by endothelial cells, such as platelet derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and angiopoietins (Angs), as well as signaling pathways involving Notch and ephrins. We will then consider the complex and multivarious contribution of pericytes to the different aspects of angiogenesis with particular emphasis on the potential role of these cells as targets in tumor therapy.

Tumor vascularity and tryptase‐positive mast cells correlate with a poor prognosis in melanoma

See commentary on page 361.

The gelatin sponge-chorioallantoic membrane assay.

Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the top of the growing CAM on day 8 of development. After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor. On day 12, blood vessels that are growing into the sponge are counted at macroscopic and microscopic levels. The estimated timeline for carrying out this protocol is 10 d. The presence of a vascular network in the CAM requires a careful analysis to distinguish new capillaries from pre-existing ones. This limitation does not occur in the avascular cornea assay, which may also take advantage of different genetic backgrounds when carried out in transgenic or knockout mice. Nevertheless, the gelatin sponge–CAM assay is simple, inexpensive and suitable for large-scale screening.*Note: In the version of the article initially published online, references 6 and 7 were incorrect. The correct references are: 6. Serbedzija, G.N., Flynn, E. &Willet, C.E. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 519–528 (2000). 7. Ribatti, D., Vacca, A., Roncali, L. &Dammacco, F. The chick embryo chorioallantoic membrane as a model for in vivo research on angiogenesis. Int. J. Dev. Biol. 40, 1189–1897 (1996). The error has been corrected in all versions of the article.

Aquaporin-4 deficiency in skeletal muscle and brain of dystrophic mdx mice

We report a detailed study of AQP4 expression in the neuromuscular system of mdx mice. Immunocytochemical analysis performed by double immunostaining revealed that mdx mice manifest a progressive reduction in AQP4 at the sarcolemmal level of skeletal muscle fast fibers and that type IIB fibers are the first to manifest this reduction in AQP4 expression. No labeling was observed in the cytoplasm of muscle fibers, indicating that the reduction in sarcolemma staining is not associated with an intracellular compartmentalization of mistargeted protein. By Western blot and RT‐PCR analysis, we found that whereas the total content of AQP4 protein decreased (by 90% in adult mdx mice), mRNA levels for AQP4 remained unchanged. A similar age related reduction in AQP4 expression was found in brain astrocytic end‐feet surrounding capillaries of mdx mice. Morphometric analysis performed after immunogold electron microscopy indicated a reduction of ~85% in gold particles (32±2/μm vs. 4.7±0.61/μm). Western blot experiments conducted using membrane fractions from brain cortex revealed a strong reduction (of 70%) in AQP4 protein in adult mdx mice, and RT‐PCR experiments demonstrated that the reduction was not at transcription level. More interesting was the finding that AQP4 reduction was associated with swelling of astrocytic perivascular processes whose ultrastructural modifications are commonly indicated as an important and early event in the development of brain edema. No apparent reduction in AQP4 was found in mdx stomach and kidney. Our data provide evidence that dystrophin deficiency in mdx mice leads to disturbances in AQP4 assembly in the plasma membrane of fast skeletal muscle fibers and brain astrocytic end‐feet, suggesting that changes in the osmotic equilibrium of the neuromuscular apparatus may be involved in the pathology of muscular dystrophy.—Frigeri, A., Nicchia, G. P., Nico, B., Quondamatteo, F., Herken, R., Roncali, L., Svelto, M. Aquaporin‐4 deficiency in skeletal muscle and brain of dystrophic mdx mice. FASEB J. 15, 90–98 (2001)

Angiogenesis spectrum in the stroma of B‐cell non‐Hodgkin's lymphomas. An immunohistochemical and ultrastructural study

Abstract: Samples of lymph nodes from 88 patients with B‐cell non‐Hodgkins lymphoma (B‐NHL) grouped by the Working Formulation (WF) and from 15 patients with benign lymphadenopathies were investigated immunohistochemically and ultrastructurally for changes in angiogenesis and stromal distribution of two subendothelial basement membrane (BM) components, namely laminin and type IV collagen. The microvessel number was usually low in lymphadenopathies, and increased significantly in low‐grade B‐NHL. Intermediate‐grade tumors displayed a further significant increase that was mainly due to their diffuse subtypes rather than to the follicular subtype. High‐grade B‐NHL showed the highest counts. By contrast with the lymphadenopathies studied, the stroma of B‐NHL reacted intensely with both BM components, whose linear co‐expression was significantly associated with low‐grade and follicular intermediate‐grade B‐NHL, while expression of laminin alone in a granular pattern was detected in diffuse intermediate‐grade and high‐grade tumors. Ultrastructural analysis revealed immature vessels more frequently in diffuse intermediate‐grade, and in high‐grade B‐NHL. These in situ data suggest that angiogenesis occurring in B‐NHL increases along their progression path, and emphasize the importance of angiogenesis as an epigenetic phenomenon of B‐NHL progression.

Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non-Hodgkin’s lymphomas

SummaryNode biopsies of 30 benign lymphadenopathies and 71 B-cell non-Hodgkin’s lymphomas (B-NHLs) were investigated for microvessel and macrophage counts using immunohistochemistry and morphometric analysis. Both counts were significantly higher in B-NHL. Moreover, when these were grouped into low-grade and high-grade lymphomas, according to the Kiel classification and Working Formulation (WF), statistically significant higher counts were found in the high-grade tumours. Immunohistochemistry and electron microscopy revealed a close spatial association between microvessels and macrophages. Overall, the results suggest that, in analogy to what has already been shown in solid tumours, angiogenesis occurring in B-NHLs increases with tumour progression, and that macrophages promote the induction of angiogenesis via the release of their angiogenic factors.

The history of the angiogenic switch concept

Spontaneously arising tumor cells are not usually angiogenic at first. The phenotypic switch to angiogenesis is usually accomplished by a substet that induces new capillaries that then converge toward the tumor. The switch clearly involves more than simple upregulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators. Tumor growth is although to require disruption of this balance and hence this switch must turned on for cancer progression. Progenitor endothelial cells, the crosstalk between angiogenic factors and their receptors and the interaction between vasculogenesis and lymphangiogenesis are all factors that may contribute to the switch. Its promotion is also the outcome of genetic instability resulting in the emergence of tumor cell lines. This review describes the history of the angiogenic switch illustrated in the literature and with particular reference to the three transgenic mouse models, namely RIP1-TAG2, keratin-14 (K14) (human papilloma virus) HPV16 and papilloma virus, used for stage-specific assessment of the effects of antiangiogenic and antitumorigenic agents.