Ultrastructure of cerebellar hemangioblastoma.

Light and ultrastructural features of a cerebellar hemangioblastoma in a 56-year-old man are described in detail. The neoplasm was composed of three major cell types: endothelial cells, pericytes, and stromal cells. The endothelial cells lined the fenestrated vascular channels. The pericytes were ensheathed by their own basal lamina which separated them from the basal lamina covering the endothelium. The stromal cells contained, in addition to the conventional organelles, numerous membrane-bound lipid inclusions, annulate lamellae, and nuclear bodies. There were also present transitional cells which shared the fine structure of all the three major cell types. Histogenetically, the tumor was considered to be of vascular origin. The stromal cells represented the stem cells which, under the neoplastic influence, continued to proliferate and differentiate into "vasoformative" elements (pericytes and endothelium) which formed new blood vessels. The transitional forms between the stromal cells and the "vasoformative" elements suggested that the cellular components of a hemangioblastoma shared a common ancestry, most likely of an angioblastic lineage.     

Angiomatosis retinae. An ultrastructural study and lipid analysis.

A nonfamilial case of agiomatosis retinae (retinal hemangioblastoma) was studied by electron microscopy. In addition to the three major types of cells previously identified within the tumor (endothelial cells, pericytes, heavily lipidized stromal cells), fibrous astrocytes in different stages of lipidization were also found. The endothelial cells were fenestrated, providing the basis for the extravasated exudate that is characteristic of the tumor. The pericytes were completely surrounded by casement membranes and displayed no significant lipidization; in a cellular plaque of vasular tissue at the base of the lesion, however, some of the multilaminar pericytes showed evidence of early smooth muscle differentiation. The stromal cells contained abundant lipid vacuoles and a few organelles, and exhibited granular degeneration of cytoplasmic filaments between the lipid vacuoles. There was spotty basement membrane formation where the stromal cells abutted on the vascular elements. No interconversion could be demonstrated among the endothelial cells, pericytes, and stromal cells. A source for the stromal cells was discovered in the early lipisization of fibrous astrocytes. Analysis of the extracted lipid from the tumor by means of infrared spectroscopy, lipid chromatography, and x-ray diffraction disclosed that the lipid was mostly cholestrol stearate, a plasma lipid. It is suggested that in the retinal lesions the leaky (fenestrated) capillaries of the tumor allowed the passive imbibition of plasma lipid by the fibrous astrocytes, leading to their gradual transformation into the fully lipidized stromal cells.     

Capillary growth: a two-cell system.

Angiogenesis is central to a number of normal and pathologic processes, including tumor growth. The identification of several angiogenic factors and the isolation and culture of capillary endothelial cells (EC) have led to a greater understanding of the cellular and biochemical bases of new vessel growth. Until recently EC have been the focus of most studies of microvascular growth. However, capillaries are not simply tubes of EC but have also a second cellular component, the mural cell or pericyte. Little is known about the later stages of vessel growth, including the addition of the pericyte to the capillary and its influence on EC growth and function. Historically the pericyte was defined by its abluminal association with the EC in the capillary. Though the pericyte's function was largely unknown, ultrastructural studies led to speculation regarding a role for the pericyte in contraction, as a stem cell and in the control of microvascular growth. Establishment of methods for the isolation, culture and identification of pericytes has permitted investigation into the role of the pericyte. EC and pericytes make frequent contact in vivo and co-culture studies of EC and pericytes reveal that the two cell types interact in a variety of ways including diffusible growth regulators, heterotypic contacts, and gap junctions. This intercellular communication is likely to be an important component of the complex mechanism(s) controlling microvascular growth and function.     

Pericytes and endothelial precursor cells: cellular interactions and contributions to malignancy

Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.     

Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis

The vasculature in the angiogenic stages of a mouse model of pancreatic islet carcinogenesis was profiled in vivo with phage libraries that display short peptides. We characterized seven peptides distinguished by their differential homing to angiogenic progenitors, solid tumors, or both. None homed appreciably to normal pancreatic islets or other organs. Five peptides selectively homed to neoplastic lesions in the pancreas and not to islet beta cell tumors growing subcutaneously, xenotransplant tumors from a human cancer cell line, or an endogenously arising squamous cell tumor of the skin. Three peptides with distinctive homing to angiogenic islets, tumors, or both colocalized with markers that identify endothelial cells or pericytes. One peptide is homologous with pro-PDGF-B, which is expressed in endothelial cells, while its receptor is expressed in pericytes.     

Differentiation and transdifferentiation potentials of cancer stem cells

Tumor cells actively contribute to constructing their own microenvironment during tumorigenesis and tumor progression. The tumor microenvironment contains multiple types of stromal cells that work together with the extracellular matrix and local and systemic factors to coordinately contribute to tumor initiation and progression. Tumor cells and their stromal compartments acquire many genetic and/or epigenetic alternations to facilitate tumor growth and metastasis. The cancer stem cell (CSC) concept has been widely applied to interpreting tumor initiation, growth, metastasis, dormancy and relapse. CSCs have differentiation abilities to generate the original lineage cells that are similar to their normal stem cell counterparts. Interestingly, recent evidence demonstrates that CSCs also have the potential to transdifferentiate into vascular endothelial cells and pericytes, indicating that CSCs can transdifferentiate into other lineage cells for promoting tumor growth and metastasis in some tissue contexts instead of only recruiting stromal cells from local or distant tissues. Although the transdifferentiation of CSCs into tumor stromal cells provides a new dimension that explains tumor heterogeneity, many aspects of CSC transdifferentiation remain elusive. In this review, we summarize the multi-lineage differentiation and transdifferentiation potentials of CSCs as well as discuss their potential contributions to tumor heterogeneity and tumor microenvironment in tumor progression.     

The role of pericytes in blood-vessel formation and maintenance

Blood vessels are composed of two interacting cell types. Endothelial cells form the inner lining of the vessel wall, and perivascular cells--referred to as pericytes, vascular smooth muscle cells or mural cells--envelop the surface of the vascular tube. Over the last decades, studies of blood vessels have concentrated mainly on the endothelial cell component, especially when the first angiogenic factors were discovered, while the interest in pericytes has lagged behind. Pericytes are, however, functionally significant; when vessels lose pericytes, they become hemorrhagic and hyperdilated, which leads to conditions such as edema, diabetic retinopathy, and even embryonic lethality. Recently, pericytes have gained new attention as functional and critical contributors to tumor angiogenesis and therefore as potential new targets for antiangiogenic therapies. Pericytes are complex. Their ontogeny is not completely understood, and they perform various functions throughout the body. This review article describes the current knowledge about the nature of pericytes and their functions during vessel growth, vessel maintenance, and pathological angiogenesis.     

The role of the microenvironment and intercellular cross-talk in tumor angiogenesis

The expression of angiogenic factors in tumors is controlled by intrinsic factors in the tumor and by the host microenvironment. Endothelial cells within particular tumor microenvironments interact not only with tumor cells but also with immune cells, fibroblasts, pericytes, and the extracellular matrix (ECM). The relationship and cross-talk among these cells determine gene expression, phenotypic distinction, and ultimately whether endothelial cells survive, proliferate, or undergo apoptosis. The diversity of angiogenic factor expression in tumors at different sites, combined with the fact that endothelial cells in different organs and tumors are phenotypically distinct, constitutes a formidable challenge for the development of effective anti-angiogenic therapies.     

Biological implications of macrophage infiltration in human tumor angiogenesis

Tumor angiogenesis is believed to be induced by increased production of angiogenic factors and decreased production of angiogenic inhibitors by cancer cells, vascular endothelial cells, and other stromal cell types. Most solid tumor cells are surrounded by stroma comprising interstitial connective tissue, blood vessels, fibroblastic cells, etc. Interaction between the stroma and malignant cells appears to have a critical role in the development of tumor neovasculature. We focused on macrophages, which demonstrate wide heterogeneity in biological function and have an essential role in tumor angiogenesis. Macrophages are terminally differentiated cells which produce a number of potent angiogenic cytokines and growth factors such as vascular endo-thelial growth factor, tumor necrosis factor-alpha, interleukin-8, and basic fibroblast growth factor. They also modulate events in the extracellular matrix through the secretion of extracellular matrix-degrading enzymes and -modulating enzymes. Thus macrophages could influence various stages of angiogenesis either positively or negatively. We found a close correlation between increased macrophage index, malignancy, and high vascular grade in malignant melanoma, and present a model for the possible involvement of activated macrophages in neovascularization in human malignant melanoma.     

Ultrastructural studies on the formation and distribution of lipid droplets in the lung capillary endothelial cells in patients with sarcoidosis

BACKGROUND AND AIM OF THE WORK: We previously reported the presence of definite morphological alterations in the capillary endothelium of sarcoid lung. The aim of this study was to examine ultrastructural changes and distribution of lipid droplets in the endothelium of lung capillaries of patients with sarcoidosis. METHODS: Tissue specimens were obtained by transbronchial lung biopsy or open lung biopsy from 16 patients with sarcoidosis and 13 controls. Biopsies were evaluated by electron microscopy following lead citrate and uranyl acetate staining. RESULTS: Typical lipid droplets were observed in pulmonary capillaries of 11 out of 16 sarcoid patients (69%); the droplet frequency was higher in sarcoid patients than in control specimens. Lipid droplets were characterized by biphasic density: most droplets contained eccentrically located vacuoles (saturated fatty acids) others were characterized by low density areas (unsaturated fatty acids). Biphasic droplets were covered by large lysosomal granules and were mainly distributed in the endothelium and pericytes. Interestingly, in the latter, vacuoles increased in size while small amounts of lysosomal granules were detectable. CONCLUSION: Our findings suggest that biphasic droplets increase in number in pulmonary capillaries of patients with sarcoidosis with a characteristic distribution pattern from the endothelium to pericytes.     

Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.     

Spontaneously formed tumorigenic hybrids of Meth A sarcoma and macrophages grow faster and are better vascularized than the parental tumor

Macrophages and Meth A sarcoma cells spontaneously fuse and give rise to tumorigenic hybrid cell lines with a mixed phenotype. We report here that the hybrid tumors grow faster and have a strikingly better developed vasculature than the parent sarcoma. Thus, electron microscopy and immunohistochemical analysis revealed that in the most active areas of neovascularization, the tumors that emerged from inocula of monoclonal hybrid cell populations had a microvessel density nearly twice that of Meth A tumors after 1 week of growth. Moreover, the proportion of vessels associated with pericytes, detected by staining for smooth muscle alpha-actin, was 3 times higher in the hybrid tumors, attesting to the more advanced differentiation of their vasculature. The collagenous stroma component was also more extensive in the hybrid tumors. Concentration of the angiogenic proteins vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were significantly higher in supernatants of hybrid cell cultures compared with Meth A cultures. These observations indicate that the growth advantage of the hybrid tumors over the parental sarcoma is due to a higher angiogenic capacity. Because the malignant features of many tumors correlate with angiogenesis and because macrophages are known to be major producers of angiogenic factors, our data open the possibility that the intense neovascularization of highly aggressive cancers in some cases reflects the acquisition of macrophage traits by heterotypic cell fusion.     

Targeting tumor vasculature with homing peptides from phage display

Tumor vasculature expresses a number of molecular markers at much lower levels than those seen in the blood vessels of normal tissues, and in some cases, such markers are undetectable. The presence of these markers relates to angiogenesis; the same markers are shared by all blood vessels undergoing angiogenesis. The endothelial cells, pericytes and smooth muscle cells, and the vascular extracellular matrix in angiogenic vessels can each express such markers. Molecularly, they represent vascular growth factor receptors, cell adhesion proteins and their receptors. Screening of phage display libraries for peptides that home to tumor vasculature when injected into mice has recently provided a new tool for analyzing the distinguishing features of tumor vasculature. Tumor-homing peptides isolated in this manner, as well as an antibody against a form of fibronectin expressed in tumor blood vessels, have been found to serve as targeting devices to concentrate drugs and other therapeutic materials to tumors in in vivo models. Such a targeting strategy can therefore potentially improve the efficacy of drugs and reduce their side effects.     

Mathematical Modelling of Angiogenesis

Angiogenesis, the formation of blood vessels from a pre-existing vasculature, is a process whereby capillary sprouts are formed in response to externally supplied chemical stimuli. The sprouts then grow and develop, driven initially by endothelial cell migration, and organize themselves into a branched, connected network. Subsequent cell proliferation near the sprout-tips permits further extension of the capillaries and ultimately completes the process. Angiogenesis occurs during embryogenesis, wound healing, arthritis and during the growth of solid tumours. In this article we first of all present a review of a variety of mathematical models which have been used to describe the formation of capillary networks and then focus on a specific recent model which uses novel mathematical modelling techniques to generate both two- and three-dimensional vascular structures. The modelling focusses on key events of angiogenesis such as the migratory response of endothelial cells to exogenous cytokines (tumour angiogenic factors, TAF) secreted by a solid tumour; endothelial cell proliferation; endothelial cell interactions with extracellular matrix macromolecules such as fibronectin; capillary sprout branching and anastomosis. Numerical simulations of the model, using parameter values based on experimental data, are presented and the theoretical structures generated by the model are compared with the morphology of actual capillary networks observed in in vivo experiments. A final conclusions section discusses the use of the mathematical model as a possible angiogenesis assay.     

The human myoepithelial cell displays a multifaceted anti-angiogenic phenotype

Human myoepithelial cells which surround ducts and acini of certain organs such as the breast form a natural border separating epithelial cells from stromal angiogenesis. Myoepithelial cell lines (HMS-1-6), derived from diverse benign myoepithelial tumors, all constitutively express high levels of active angiogenic inhibitors which include TIMP-1, thrombospondin-1 and soluble bFGF receptors but very low levels of angiogenic factors. These myoepithelial cell lines inhibit endothelial cell chemotaxis and proliferation. These myoepithelial cell lines sense hypoxia, respond to low O2 tension by increased HIF-1 alpha but with only a minimal increase in VEGF and iNOS steady state mRNA levels. Their corresponding xenografts (HMS-X-6X) grow very slowly compared to their non-myoepithelial carcinomatous counterparts and accumulate an abundant extracellular matrix devoid of angiogenesis but containing bound angiogenic inhibitors. These myoepithelial xenografts exhibit only minimal hypoxia but extensive necrosis in comparison to their non-myoepithelial xenograft counterparts. These former xenografts inhibit local and systemic tumor-induced angiogenesis and metastasis presumably from their matrix-bound and released circulating angiogenic inhibitors. These observations collectively support the hypothesis that the human myoepithelial cell (even when transformed) is a natural suppressor of angiogenesis. Oncogene (2000) 19, 3449 - 3459     

Phenoxodiol, an experimental anticancer drug, shows potent antiangiogenic properties in addition to its antitumour effects

Phenoxodiol (2H-1-benzopyran-7-0,1, 3-[4-hydroxyphenyl], PXD) is a synthetic analogue of the naturally-occurring plant isoflavone and anticancer agent, genistein. PXD directly induces mitotic arrest and apoptosis in most cancer cells and is currently undergoing clinical trials, as a chemotherapeutic in ovarian and prostate cancers. We show here that PXD also exhibits potent antiangiogenic properties. Thus, it inhibited endothelial cell proliferation, migration and capillary tube formation and inhibited expression of the matrix metalloproteinase MMP-2, a major matrix degrading enzyme. Importantly, we demonstrate that PXD is functional in vivo since it inhibited the extent of capillary tube invasion in an in vivo model of angiogenesis. We show that phenoxodiol inhibits hallmarks of endothelial cell activation, namely TNF or IL-1 induced E-selectin and VCAM-1 expression and IL-8 secretion. However, PXD had no effect on unstimulated endothelial cells. We also describe that PXD inhibits the lipid kinase sphingosine kinase, which recently has been implicated in endothelial cell activation and angiogenesis as well as oncogenesis. Thus, our results suggest that PXD may be an effective anticancer drug targeting the two drivers of tumour growth--the proliferation of the tumour cells themselves and the angiogenic and inflammatory stimulation of the vasculature.     

Bone marrow angiogenesis in multiple myeloma.

Angiogenesis is a constant hallmark of multiple myeloma (MM) progression and has prognostic potential. It is induced by plasma cells via angiogenic factors with the transition from monoclonal gammopathy of undetermined significance (MGUS) to MM, and probably with loss of angiostatic activity on the part of MGUS. The pathophysiology of MM-induced angiogenesis is complex and involves both direct production of angiogenic cytokines by plasma cells and their induction within the microenvironment. The latter are secreted by stromal cells, endothelial cells (EC) and osteoclasts, and promote plasma cell growth, survival and migration, as well as paracrine cytokine secretion and angiogenesis in the bone marrow milieu. Angiogenesis is also supported by inflammatory cells following their recruitment and activation by plasma cells. Finally, circulating EC and endothelial precursor cells (EPC) contribute to the neovascularization, and the presence of EPC suggests that vasculogenesis (new vessel formation from EPC) may also contribute to the full MM vascular tree.     

Angiogenic activity of mesenchymal stem cells in multiple myeloma

The pathophysiology of multiple myeloma-induced angiogenesis is complex and involves both direct production of angiogenic cytokines by plasma cells and their induction within the microenvironment. In this research, we investigated whether mesenchymal stem cells participated in inducing the angiogenic response in multiple myeloma, and explored the mechanism by which MSCs influence myeloma angiogenesis. We detected the concentration of angiogenic factors (bFGF, HGF, and VEGF) in the conditioned medium of mesenchymal stem cells and the capillary formation ability of mesenchymal stem cells in vitro. We found that conditioned medium of MSCs derived from MM significantly promoted the proliferation, chemotaxis, and capillary formation of human umbilical vein endothelial cells compared with that from normal donors. ELISA and RT-PCR were used to detect the mRNA and protein levels of angiogenic factors (bFGF, HGF, and VEGF) in the conditioned medium. We found that mRNA and protein levels of angiogenic factors were elevated in MSCs from multiple myeloma compared with normal donors.     

糖尿病大鼠视网膜Müller细胞的超微结构变化

目的研究糖尿病大鼠视网膜Müller细胞超微结构变化特点及其在糖尿病视网膜病变的作用。方法应用透射电镜技术系统观察糖尿病大鼠4w、8w、12w和24w视网膜病理改变,特别是视网膜Müller细胞形态变化特点。结果视网膜Müller细胞从4w开始出现少量线粒体肿胀,嵴溶解,并随糖尿病的病程逐渐加重,线粒体肿胀、嵴断裂、空泡状明显。各时段均未发现典型的毛细血管闭锁、管腔狭窄等改变,血管内皮细胞、周细胞基本正常。结论糖尿病视网膜Müller细胞的超微结构改变要早于视网膜血管内皮细胞及周细胞超微结构的改变,在神经节细胞层及神经纤维层改变最明显,并随糖尿病时间的延长Müller细胞形态结构改变更加明显。     

MT1-MMP: Universal or particular player in angiogenesis?

Tumorigenesis involves not only tumor cells that become transformed but also the peritumoral stroma which reacts inducing inflammatory and angiogenic responses. Angiogenesis, the formation of new capillaries from preexisting vessels, is an absolute requirement for tumor growth and metastasis, and it can be induced and modulated by a wide variety of soluble factors. During angiogenesis, quiescent endothelial cells are activated and they initiate migration by degrading the basement membranes through the action of specific proteases, in particular of matrix metalloproteinases (MMPs). Among these, the membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified as a key player during the angiogenic response. In this review, we will summarize the role of MT1-MMP in angiogenesis and the regulatory mechanisms of this protease in endothelial cells. Since our recent findings have suggested that MT1-MMP is not universally required for angiogenesis, we hypothesize that the regulation and participation of MT1-MMP in angiogenesis may depend on the nature of the angiogenic stimulus. Experiments aimed at testing this hypothesis have shown that similarly to the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12, lipopolysaccharide (LPS) seems to induce the formation of capillary tubes by human or mouse endothelial cells (ECs) in an MT1-MMP-independent manner. The implications of these findings in the potential use of MT1-MMP inhibitors in cancer therapy are discussed.