Loss of RhoA promotes skin tumor formation and invasion by upregulation of RhoB.

Cellular movement is controlled by small GTPases, such as RhoA. Although migration is crucial for cancer cell invasion, the specific role of RhoA in tumor formation is unclear. Inducing skin tumors in mice with a keratinocyte-restricted loss of RhoA, we observed increased tumor frequency, growth and invasion. In vitro invasion assays revealed that in the absence of RhoA cell invasiveness is increased in a Rho-associated protein kinase (ROCK) activation and cell contraction-dependent manner. Surprisingly, loss of RhoA causes increased Rho signaling via overcompensation by RhoB because of reduced lysosomal degradation of RhoB in Gamma-aminobutyric acid receptor-associated protein (GABARAP)+ autophagosomes and endosomes. In the absence of RhoA, RhoB relocalized to the plasma membrane and functionally replaced RhoA with respect to invasion, clonogenic growth and survival. Our data demonstrate for the first time that RhoA is a tumor suppressor in 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol 13-acetate skin carcinogenesis and identify Rho signaling dependent on RhoA and RhoB as a potent driver of tumor progression.

Changes in the electrical properties of the electrode-skin-underlying tissue composite during a week-long programme of neuromuscular electrical stimulation.

Particular neuromuscular electrical stimulation (NMES) applications require the use of the same electrodes over a long duration (>1 day) without having access to them. Under such circumstance the quality of the electrode-skin contact cannot be assessed. We used the NMES signal itself to assess the quality of the electrode-skin contact and the electrical properties of the underlying tissues over a week. A 14% decrease in the skin's stratum corneum resistance (from 20 to 17 kΩ) and a 15% decrease in the resistance of the electrodes and underlying tissues (from 550 to 460 Ω) were observed in the 14 healthy subjects investigated. A follow-on investigation of the effect of exercise-induced sweating on the electrical properties of the electrode-skin-underlying tissue composite during NMES indicated a correlation between the decrease in the resistance values observed over the course of the week and the accumulation of sweat at the electrode-skin interface. The value of the capacitance representing the dielectric properties of the skin's stratum corneum increased after exercise-induced sweating but did not change significantly over the course of the week. We conclude that valuable information about the electrode-skin-underlying tissue composite can be gathered using the NMES signal itself, and suggest that this is a practical, safe and relatively simple method for monitoring these electrical properties during long-term stimulation.

Expression of glutathione S-transferase T1 (GSTT1) in human brain tumours.

Glutathione S-transferases (GSTs) play a central role in a number of metabolic processes. Glutathione S-transferase T1 (GSTT1) is a polymorphic cytosolic enzyme and a member of the theta class of GSTs. Typical substrates for GSTT1 are industrial compounds, such as dichloromethane and ethylene oxide. It has been shown that also chemotherapeutic drugs such as BCNU [i.e. 1,3-bis(2-chloroethyl)-1-nitrosourea] are efficiently inactivated by GSTT1. BCNU is a drug which is increasingly used locally in the chemotherapy of glioblastoma multiforme WHO grade IV. Therefore, if GSTT1 were expressed in neoplastic cells of brain tumours it could be a factor for chemoresistance. In order to clarify a possible role of GSTT1 in chemoresistance, as a first step, we localized this enzyme in malignant gliomas such as glioblastoma multiforme WHO grade IV and oligodendroglioma WHO grade II. Because of its polymorphism we first genotyped the samples for GSTT1 by PCR. Using in situ hybridization, we then demonstrated that GSTT1 transcripts are expressed in neoplastic cells of both tumour types. Immunohistochemistry revealed then that whereas neoplastic cells in glioblastoma multiforme WHO grade IV contain GSTT1, it was not localized in oligodendroglioma cells. Given the polymorphism of GSTT1 and its potential activity towards BCNU, the localization of GSTT1 in glioblastoma cells can be considered as a possible factor of non-homogeneous chemotherapy response among patients with different GSTT1 genotypes.

Apoptosome inactivation rescues proneural and neural cells from neurodegeneration.

Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in Apaf1(-/-) embryos escape programmed cell death, proliferate and retain their potential to differentiate. To evaluate the circumstances of Apaf1(-/-) NPC survival and investigate their fate under neurodegenerative conditions, we established cell lines of embryonic origin (ETNA). We found that Apaf1(-/-) NPCs resist common apoptotic stimuli and neurodegenerative inducers such as amyloid-beta peptide (typical of Alzheimer's disease) and mutant G93A superoxide dismutase 1 (typical of familial amyotrophic lateral sclerosis). Similar results were obtained in Apaf1(-/-) primary cells. When death is prevented by Apaf1 deficiency, cytochrome c is released from mitochondria and rapidly degraded by the proteasome, but mitochondria remain intact. Under these conditions, neither activation by cleavage of initiator caspases nor release of alternative apoptotic inducers from mitochondria takes place. In addition, NPCs can still differentiate, as revealed by neurite outgrowth and expression of differentiation markers. Our findings imply that the mitochondrion/apoptosome pathway is the main route of proneural and neural cells to death and that its inhibition prevents them from dismantling in neurodegenerative conditions. Indeed, the ETNA cell model is ideally suited for exploring the potential of novel cell therapies for the treatment of human neurodegenerations.

Ultrastructural localization of integrin subunits alpha3 and alpha6 in capillarized sinusoids of the human cirrhotic liver.

Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.

Glycoconjugate distribution in early human notochord and axial mesenchyme.

Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier.

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy. In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier disruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes. All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.

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 approximately 85% in gold particles (32+/-2/microm vs. 4.7+/-0.61/microm). 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.

Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes.

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.

Extensive glycosylation changes revealed by lectin histochemistry in morphologically normal prenatal tissues of the mouse mutant undulated (un/un).

Recently we observed that in human embryos and fetuses with a variety of malformations, not only malformed tissues, but also several non-malformed tissues displayed alterations in the glycosylation pattern. It was the aim of this work to investigate this more or less inexplicable phenomenon under experimental conditions. To this end, we studied a well known mouse model, the mouse mutant undulated, which has an exactly defined genetic defect (substitution in the pax-1 gene) leading to a localized malformation in the vertebral column. The glycosylation pattern was studied using lectin histochemistry. Distribution of binding sites for the lectins RCA I, Con A, SNA, SBA, PNA, LTA and WGA was studied during the organogenesis stages (i.e., days 11-18). It was striking that in mutants, changes in the glycosylation pattern were found not only in the malformed organ (i.e., vertebral anlage), but also in other embryonic tissues, which showed normal morphology. This suggests that the altered glycosylation seems to be a part of genetically determined phenomena throughout the entire organism. Our results show that a defect in a gene with a very restricted expression can cause universal changes in the glycosylation pattern during development.

Interferon beta-1a prevents the effects of lipopolysaccharide on embryonic brain microvessels.

By means of light and electron microscopy we have studied the effect of interferon beta-1a (IFNbeta-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood-brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for beta-D-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNbeta-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNbeta-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNbeta-1a.

Nidogen-1. Expression and ultrastructural localization during the onset of mesoderm formation in the early mouse embryo.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)

Localization of cathepsin D in human odontoclasts. a light and electron microscopical immunocytochemical study.

Odontoclasts are dentine and cementum resorbing cells whose relationship to bone resorbing osteoclasts is not clear. Like osteoclasts, they possess different cathepsins which are involved in mineralized tissue degradation during the tooth root resorption process in deciduous teeth. Whether cathepsin D, which in osteoclasts probably functions as an activator of other cathepsins, can be found in odontoclasts, has, however, not been investigated before. In order to determine its occurrence and localization, cathepsin D immunocytochemistry was applied to paraffin-embedded sections from 30 human deciduous tooth roots undergoing resorption. Using immunogold postembedding immunocytochemsitry on LR-Gold embedded specimens, the distribution of cathepsin D was investigated at the ultrastructural level. We identified tartrate-resistent acid phosphatase-positive mono- and multinuclear odontoclasts near and on the periodontal surfaces of tooth roots. Nearly all of these cells showed cytoplasmic granular cathepsin D immunoreactivity. At the electron microscopical level, gold labelling was seen on vacuoles and vesicles of the odontoclasts, which were identified as secondary lysosomes and phagosomes. Extracellularly it was seen along the ruffled border and in neighboured resorption areas of dentine and cementum. These findings indicate that cathepsin D is secreted into the resorbing area of human odontoclasts in order to participate in degradation of mineralized tooth matrix, but may also function as an activator of other proteases in lysosomal organelles.

Matrix metalloproteinases in early human liver development.

Matrix metalloproteinases (MMPs) regulate matrix deposition in tissues. Collagens I, III, and IV are involved in early human liver development. To establish whether MMPs specific for these collagens participate in early human liver development, we localized immunohistochemically MMP-1 and MMP-13 (for collagens I and III) and MMP-2 and MMP-7 (for collagen IV) in the early human liver anlage [6th-10th gestational week (GW)]. MMP-1 was found from the 6th GW onward in hepatocytes and later also in outer limiting plate hepatocytes, early bile ducts, and periportal mesenchymal cells. In the 6th GW, MMP-2 was found only in microvascular endothelium. In the 7th GW, MMP-2 was also detected in hepatocytes. From the 9th GW onward, MMP-2 was detectable in all hepatocytes and erythropoietic, endothelial, and periportal mesenchymal cells. MMP-7 was present in the 6th GW in some hepatocytes and endothelial cells, but from the 7th GW onward, only in hematopoietic cells. MMP-13 was found exclusively in hematopoietic cells. This study has shown that production of MMP -1, MMP-2, MMP-7, and MMP-13 during human liver development already occurs from the 6th GW. At this time-point their substrates are only traces or are not yet present in the tissue. A possible role of MMPs in early liver development is discussed.

Transforming growth factor-beta1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells.

BACKGROUND/AIMS: It is suggested that during fibrogenesis as well as during carcinogenesis of the liver, the hepatic microvascular phenotype is transformed from sinusoids - which lack a basement membrane--into continuous capillaries which rest on a basement membrane. As transforming growth factor (TGF)-beta1 seems to be the most effective mediator in the stimulation of matrix protein synthesis, we were interested in the modulation of basement membrane proteins collagen type IV, laminin, and entactin expression by TGF-beta1 in liver sinusoidal endothelial cells (SECs), especially since a stimulation of the synthesis of collagen type IV but not of entactin and laminin by TGF-beta1 has been demonstrated in a fibrosarcoma cell line. METHODS: The synthesis of the basement membrane (BM) proteins entactin, laminin, and collagen type IV and of the extracellular matrix (ECM) proteins tenascin and fibronectin with or without TGF-beta1--stimulation was analyzed by immunostaining, immunoprecipitation of endogenously labeled proteins and Northern blot analysis of total RNA extracted from freshly isolated or cultured SECs from rat or guinea pig livers. Furthermore, SECs were isolated from acutely and chronically CCl4-damaged rat livers and were analyzed for matrix protein expression. RESULTS: SECs were adherent 24 h after isolation and formed confluent monolayers on day 4 of primary culture. Specific immunoprecipitates and specific transcripts for the BM proteins entactin, laminin, and collagen type IV and for ECM proteins tenascin and fibronectin were detectable in freshly isolated or cultured SECs. The synthesis of all tested BM proteins and ECM proteins was stimulated at least 3-fold by TGF-beta1. In SECs isolated after CCl4-induced acute and chronic liver damage, increased levels of matrix protein transcripts were detectable. CONCLUSIONS: The stimulation of the synthesis of all BM-proteins by TGF-beta1 in vitro and the accumulation of ECM transcripts in SECs isolated from CCl4-treated livers, suggests that SECs are involved in the formation of a basement membrane during the "capillarization" of the sinusoids during liver disease.

Developmental expression of ZO-1 antigen in the mouse blood-brain barrier.

Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.

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

Node 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.