Luisa Roncali

Bone marrow angiogenesis and progression in multiple myeloma.

Tumour growth is angiogenesis‐dependent. We found a high correlation between the extent of bone marrow angiogenesis, evaluated as microvessel area, and the proliferating (S‐phase) fraction of marrow plasma cells, evaluated as labelling index (LI), in patients with multiple myeloma (MM) and in those with monoclonal gammopathies of undetermined significance (MGUS). Angiogenesis itself was significantly associated with active as opposed to non‐active MM and MGUS. The highest microvessel area accompanied rapidly progressive MM with the highest LI. When a cut‐off value of 2% or greater of the microvessel area was used, most patients with active MM were classified correctly. The risk of active disease in patients with MM increased in parallel with the microvessel area. A causal relationship between plasma cell growth, activity phase in MM and marrow angiogenesis is suggested. Since angiogenesis proceeds in step with the enlargement of plasma cell tumours and the activity phase in MM, its measurement could be a useful prognostic marker in patients with plasma cell proliferative disorders.

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.

New Model for the Study of Angiogenesis and Antiangiogenesis in the Chick Embryo Chorioallantoic Membrane: The Gelatin Sponge/ Chorioallantoic Membrane Assay

Several methods for the in vivo study of angiogenesis are available, and each angiogenic assay presents distinct advantages and disadvantages. In this study, we present a new method for the quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM), based on the implantation of gelatin sponges on the top of growing CAM, on day 8 of incubation. After implantation, the sponges were treated with a stimulator (recombinant human basic fibroblast growth factor, FGF2) or an inhibitor (a rabbit polyclonal anti-FGF2 antibody) of blood vessel formation. Blood vessels growing vertically into the sponge and at the boundary between sponge and surrounding CAM mesenchyme were counted by a morphometric method on day 12. In addition, to assess whether the gelatin sponge is an appropriate vehicle to deliver cultured cells and evaluate their angiogenic potential, mouse aortic endothelial cells were cotransfected with human FGF2 and the Escherichia coli beta-galactosidase (beta-GAL) reporter gene. Stable transfectants were absorbed by the sponge, and evaluation of the angiogenic response was paralleled by beta-GAL staining to visualize implanted cells. This technique may facilitate the discovery and development of agonists or antagonists of angiogenesis.

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.

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 and mast cell density with tryptase activity increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas.

Node biopsies of 16 benign lymphadenopathies and 72 B‐cell non‐Hodgkins lymphomas (B‐NHLs) were investigated for counts of microvessels, total metachromatic mast cells (MCs) and MCs expressing tryptase, an angiogenesis‐inducing molecule. Counts were higher in B‐NHLs. When grouped according to the Working Formulation (WF) malignancy grades, they were significantly higher in low‐grade B‐NHLs vs. lymphadenopathies and intermediate‐grade vs. low‐grade tumors and there was a further increase in the high‐grade tumors. A high correlation was demonstrated in all groups of tissues between microvessel counts and both total metachromatic and tryptase‐reactive MCs. These results suggest that angiogenesis in B‐NHLs increases with their progression, and that MCs cooperate in its induction via the tryptase contained in their secretory granules. Int. J. Cancer 85:171–175, 2000. ©2000 Wiley‐Liss, Inc.

Severe Alterations of Endothelial and Glial Cells in the Blood-Brain Barrier of Dystrophic mdx Mice

In this study, we investigated the involvement of the blood‐brain barrier (BBB) in the brain of the dystrophin‐deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO‐1) and claudin‐1 proteins, and a glial marker, the aquaporin‐4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron‐dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO‐1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO‐1 immunocytochemistry and claudin‐1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO‐1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO‐1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of α‐actin and ZO‐1 revealed colocalization of these proteins. As in ZO‐1 staining, the pattern of immunolabeling with anti–α‐actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. α‐actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas α‐actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti‐AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4‐, CD8‐, CD20‐, and CD68‐positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in α‐actin cytoskeleton, ZO‐1, claudin‐1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD. GLIA 42:235–251, 2003.

Endothelial Cell Heterogeneity and Organ Specificity

Endothelial cells consist of a heterogeneous population covering the entire inner surface of blood vessels. This review will focus on the factors influencing this heterogeneity including: (1) morphological and functional differences between large and small vessels and between cells derived from various microvascular endothelial beds; (2) the microenvironment and extracellular matrix modulating the phenotype; (3) different response to growth factors; (4) organ specificity reflecting the cumulative expression of post-translation modifications and also the expression of unique genes under the control of organ-specific regulatory elements; and (5) pathological conditions, such as tumor growth, which is accompanied by the development of a characteristic tumor vasculature and tumors formed by endothelial cells.

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis

In order to better understand the process of angiogenesis in the developing human brain, we have examined the spatial relationship and relative contributions of endothelial cells and pericytes, the two primary cell types involved in vessel growth, together with their relation with the vascular basement membrane. Pericytes were immunolocalized through use of the specific markers nerve/glial antigen 2 (NG2) proteoglycan, endosialin (CD248) and the platelet-derived growth factor receptor β (PDGFR-β), while endothelial cells were identified by the pan-endothelial marker CD31 and the blood brain barrier (BBB)-specific markers claudin-5 and glucose transporter isoform 1 (GLUT-1). The quantitative analysis demonstrates that microvessels of the fetal human telencephalon are characterized by a continuous layer of activated/angiogenic NG2 pericytes, which tightly invest endothelial cells and participate in the earliest stages of vessel growth. Immunolabelling with anti-active matrix metalloproteinase-2 (aMMP-2) and anti-collagen type IV antibodies revealed that aMMP-2 producing endothelial cells and pericytes are both associated with the vascular basement membrane during vessel sprouting. Detailed localization of the two vascular cell types during angiogenesis suggests that growing microvessels of the human telencephalon are formed by a pericyte-driven angiogenic process in which the endothelial cells are preceded and guided by migrating pericytes during organization of the growing vessel wall.