Expression of VEGFR-3 and 5'-nase in regenerating lymphatic vessels of the cutaneous wound healing

The vascular endothelial growth factor-C (VEGF-C), a specific lymphangiogenic growth factor, raises new questions and perspectives in studying lymphatic development and regeneration. Wound healing skins in mice were processed for 5'-nucleotidase (5'-Nase) and VEGFR-3 (the receptor of VEGF-C) histochemical staining to distinguish lymphatics from blood capillaries and to analyze lymphangiogenesis. In the wounds of 3-5 days after injury, anti-VEGFR-3 immunopositive signals unevenly appeared in 5'-Nase-positive lymphatic vessels in the subcutaneous tissue. A few small circular and irregular lymphatic-like structures with VEGFR-3 expression scattered in the dermal and subcutaneous tissues. Between days 7 and 15 of the wounds, numerous accumulated vasculatures were stained for 5'-Nase and PECAM-1, extending irregularly along the wound edge. Von Willebrand factor was expressed in the endothelial cells of blood vessels and lymphatics in the subcutaneous tissue. Ultrastructural changes of lymphatic vessels developed at different stages, from lymphatic-like structures to newly formed lymphatic vessels with an extremely thin and indented wall. Endothelial cells of the lymphatic vessel were eventually featured by typical intercellular junctions, which deposited with reaction products of VEGFR-3 and 5'-Nase-cerium but lacked VEGF-C expression. The present findings indicate that VEGF-C-induced lymphangiogenesis occurs from the subcutaneous to the dermis along the wound healing edge, especially in the dermal-subcutaneous transitional area, favorable to growth of regenerating lymphatic vessels.     

Development of the avian lymphatic system

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.     

Lymphangiogenesis in tumors: what do we know?

Lymphangiogenesis, the growth of new lymphatic vessels, has long been regarded as a putative efficient pathway to neoplastic metastization. However, until recently consistent data regarding reliable lymphatic endothelial cell markers were lacking. Moreover, the presence of new formed lymphatic vessels was considered a largely disputable concept. Now, this scenario has changed significantly, owing to consistent reports describing novel lymphatic endothelial cell (LEC) markers, the demonstration of new formed lymphatic vessels within the bulk of the tumor in animal models and human neoplasms, and the characterization of the VEGF-C/VEGFR-3 pathway. We herein review the major breakthroughs in the field of lymphangiogenesis, with special emphasis on novel and reliable LEC markers, such as prox-1, LYVE-1, and podoplanin, as well as on the pathological assessment of lymphangiogenesis as a putative prognostic factor for human neoplasms.     

Lymphatic function, lymphangiogenesis, and cancer metastasis

The lymphatic system serves as the primary route for the metastasis of many cancers and the extent of lymph node involvement is the most important indicator of tumor aggressiveness. Despite the apparent importance of the lymphatic vessels for tumor dissemination, it has remained unclear whether activation of lymphatic endothelial cells may affect tumor progression and metastasis and the molecular mechanisms of lymphangiogenesis are just beginning to be elucidated. This overview describes the unique structural and functional characteristics of the lymphatic vessels that render them particularly suitable for invasion by tumor cells and for their efficient transport to lymph nodes. Recent evidence indicates occurrence of tumor lymphangiogenesis and its correlation with metastasis. Molecular regulation of tumor lymphangiogenesis, its significance for tumor metastasis, and implications for cancer therapy are discussed.     

Role of microglia in glioma biology.

Microglia, a type of differentiated tissue macrophage, are considered to be the most plastic cell population of the central nervous system (CNS). In response to pathological conditions, resting microglia undergo a stereotypic activation process and become capable of phagocytosis, antigen presentation, and lymphocyte activation. Considering their immune effector function, it is not surprising to see microglia accumulation in almost every CNS disease process, including malignant brain tumors or malignant gliomas. Although the function of these cells in CNS inflammatory processes is being studied, their role in malignant glioma biology remains unclear. On one hand, microglia may represent a CNS anti-tumor response, which is inactivated by local secretion of immunosuppressive factors by glioma cells. On the other hand, taking into account that microglia are capable of secreting a variety of immunomodulatory cytokines, it is possible that they are attracted by gliomas to promote tumor growth. A better understanding of microglia-glioma interaction will be helpful in designing novel immune-based therapies against these fatal tumors.     

Histochemical analysis of lymphatic endothelial cells in lymphostasis

The ultrastructure of endothelial cells of intestinal lymphatics and the thoracic duct (TD) and the relation to lymphostasis were examined in rats and monkeys. Localization of 5'-nucleotidase (5'-Nase) and endothelial nitric oxide synthase (eNOS) was studied. In normal lymphatic endothelial cells, 5'-Nase reaction product was evenly deposited on the cell surface in vivo and on cultured TD endothelial cells (TDECs), whereas eNOS was evenly distributed throughout the nucleus and cytoplasm. TDECs had a long filamentous process extending towards the subendothelial extracellular matrix but became flat and regular within 30-40 minutes after gastric perfusion with olive oil. According to their electron-density, two types of cells were found in the TD endothelial layer. The cells with low electron-density exhibited stronger 5'-Nase activity. Valves were bicuspid formations and the valvular endothelial surface of the convex side showed weaker 5'-Nase activity than the concave side. During TD blockage-induced lymphostasis in rats, the 5'-Nase product was almost not discernible in the TDECs within 2 weeks. Larger vesicles were found in the endothelial cytoplasm of the ligated TD. Their number decreased after 6-12 weeks. The small intestinal lymphatics in the mucosa and submucosa were dilated, with numerous open intercellular junctions. The endothelial lining appeared to have reduced activities for 5'-Nase and eNOS in 9 of 11 experimental animals. The results indicated that the inability of the open intercellular junctions, normally working as one-way endothelial flap valves, may be a key morphological feature after TD blockage. Reduced eNOS and 5'-Nase may functionally influence contractile activity and transport capability of the lymphatic vessels in the lymphostasis.     

Angiogenesis in brain tumors

Angiogenesis is a complex process regulated by multiple stimulatory and inhibitory factors that are able to modulate the migration and/or proliferation of microvascular cells with the objective of formation of neovasculature from preexisting vessels. It involves well-coordinated steps including production and release of angiogenic factors, proteolytic degradation of extracellular matrix components to allow formation of capillary sprout, proliferation and directional migration of microvascular cells, and the final composition of new vessels [Senger (1996) Am. J. Pathol. 149:1-7]. Angiogenesis is present in a number of hypoxic and/or ischemic conditions in the central nervous system, in particular in infarctions and infectious processes. Angiogenesis also plays an important role in malignant primary tumors. Glioblastomas, the most malignant gliomas in adults, are among the most angiogenic of all human tumors. This review will examine recent data of the role of angiogenic growth factors in the neoplastic and reactive conditions in the brain.     

Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix

Angiogenesis is critical to wound repair. Newly formed blood vessels participate in provisional granulation tissue formation and provide nutrition and oxygen to growing tissues. In addition, inflammatory cells require the interaction with and transmigration through the endothelial basement membrane to enter the site of injury. Angiogenesis, in response to tissue injury, is a dynamic process that is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment. Vascular endothelial growth factor, angiopoietin, fibroblast growth factor, and transforming growth factor beta are among those most potent angiogenic cytokines in wound angiogenesis. The cooperative regulation of them is essential for wound repair. Migration of endothelial cells and development of new capillary vessels during wound repair is dependent on not only the cells and cytokines present but also the production and organization of ECM components both in granulation tissue and in endothelial basement membrane. The ECM regulates angiogenesis by providing scaffold support and signaling roles. They also serve as a reservoir and modulator for growth factors. Laminins are the major noncollagenous ECM of endothelial basement membrane. Two newly recognized laminins, 8 and 10, are the major laminins produced by human dermal microvascular endothelial cells. Laminin 10 is highly expressed in blood vessels around skin wounds. Laminin 8 promotes dermal endothelial cell attachment, migration, and tubule formation. Integrins with either beta 1 or alpha v subunits are the major cellular surface receptors for ECM molecules and mediate the interactions between cells and ECM during wound angiogenesis.     

Tumor angiogenesis of non-small cell lung cancer

Lung cancer is one of the commonest causes of cancer death in developed countries. Recent evidence suggests that angoigenesis is related to poor prognosis in many solid tumors including non-small cell lung cancer (NSCLC). Angiogenesis is regulated by a complex interaction among growth factors and cytokines and influenced by proteolytic enzymes such as plasminogen activators and matrix metalloproteases, expression of adhesion molecules, and distribution of extracellular matrices. Fibroblasts, macrophages, mast cells, and endothelial cells themselves also affect angiogenesis. This review concentrates on angiogenic growth factors including vascular endothelial growth factor, angiopoietins, platelet derived endothelial growth factor, and basic fibroblast growth factor, proteases, adhesion molecules including vascular endothelial cadherin and integrins, osteopontin, and mast cell products in tumor angiogenesis of NSCLC.     

Angiogenesis in normal and neoplastic pituitary tissues

Angiogenesis, or the formation of new blood vessels, is a dynamic process needed for embryogenesis, post-natal growth, morphogenesis, tumorigenesis, and for other biological processes. Angiogenesis is very important for tumor development and progression. This review examines the activators and inhibitors of angiogenesis with emphasis on the pituitary gland and pituitary neoplasms. Some of the proteins regulating angiogenesis in pituitary tumors such as vascular endothelial growth factor (VEGF) and VEGF receptors, fibroblasts growth factors (FGF), transforming growth factor beta (TGFB), interleukins, interferons, and matrix metalloproteinases (MMPs) and inhibitors of MMPs have been examined in animal and human pituitary tumor models. However, many other significant regulators of angiogenesis including angiopoietins, angiostatin, and thrombospondins have not been studied extensively in pituitary tumors to date. Newer concepts and developments in angiogenesis such as vasculogenic mimicry and gene therapy approaches to angiogenesis in cancer treatment are also discussed.     

The antitumor effects of Newcastle disease virus on glioma

Glioma is the most common primary malignant brain tumor with a poor survival rate. In recent years, no significant progress has been made in the treatment of gliomas in contrast to the development of improved diagnosis via molecular typing. Newcastle disease virus (NDV), a negative-stranded RNA virus that exhibits oncolytic activity, has been investigated for its capacity to elicit antitumor activity in many types of cancers, including glioma. Therefore, application of oncolytic viruses, such as NDV, as a new treatment strategy to specifically target aberrant signaling in glioblastomas has brought new hope. For many years, NDV has been investigated for its in vivo and in vitro efficacy in the treatment of various tumor cells. Based on its safety in humans, specificity for tumor cells, and immunostimulatory properties, NDV represents a promising antitumor agent. In this review, we summarize the background of NDV and the antitumor mechanisms of NDV-mediated oncolysis, discuss the potential value and role of NDV in gliomas, and describe new advances and perspectives for future research.     

Adhesion of endothelial cells using self-assembly peptides under precise deformation control of tissue-engineered vessels

The objective of this study was to anchor endothelial cells using self-assembly peptides under precise deformation control of tissue-engineered vessels. An acelluarized vascular matrix was used as the control group to examine the function of self-assembly peptides. In the experiment group, the self-assembly peptides were added to the inner surface of tissue-engineered vessels to form a monolayer. Then the endothelial cells were injected into the vascular lumen. A deformation control system was developed which was based on real-time image analysis and feedback control system. After dynamic culture by different deformation (set points 1, 5, and 10 per cent), the endothelial cell densities of experimental and control groups were compared. Both the self-assembly peptides and the extent of deformation affected the endothelial cell density on the inner surface of tissue-engineered vessels. The construct with self-assembly peptides under 5 per cent deformation gained the highest endothelial cell density. It was concluded that the deformation of assembled peptides contributes to the development and adhesion of endothelial cells in the inner surface of tissue-engineered vessels.     

Embryonic erythropoiesis in human yolk sac: two different compartments for two different processes

The wall of 12 yolk sacs (YSs) from 17- to 50-day-old human embryos was examined by light, scanning, and transmission electron microscopy to identify the ontogeny of embryonic erythropoiesis. Initial formation of blood island with the generation of erythroid and endothelial cells was seen in the mesenchymal layer in embryos aged 17 days. A network of blood vessels containing abundant erythroblasts was identified in the YS walls of embryos aged approximately 24 days. At this age, erythroblasts were also identified within the embryo body. Primitive erythroblasts were the only cells present within the embryo and its YS until the end of week 5. These cells first appeared in the mesenchymal vascular plexus of the YS wall, and were then observed in the liver and other tissues of the embryo. At embryonic week 5, two compartments were identified in the YS wall; a mesodermal one in which blood vessels were formed, and an endodermal compartment in which erythrocytes were present within the endodermal vesicles. Erythrocytes were small non-nucleated cells similar to adult erythrocytes. Transmission electron microscopic observation focused on the endodermal vesicles confirmed the presence of definitive erythrocytes only at such extra vascular location. At this age, there were no definitive erythrocytes detected within the embryo. Erythrocytes started to be identified in embryonic blood vessels from week 7 onward. These findings provide information not previously described about YS erythropoiesis during early human development.     

Formation of a functional endothelium on vascular grafts

The lack of a functional endothelial cell lining on artificial polymeric vascular grafts severely reduces their effectiveness in replacing small caliber (< 6 mm) blood vessels. Techniques have now been developed to transplant autologous endothelial cells from one site in the body onto the surface of grafts prior to implantation. Pre-clinical animal trials provide evidence that grafts sodded with autologous, fat-derived, microvessel endothelial cells exhibit a stable, antithrombogenic lining of endothelium. The new endothelial cell lining exhibits morphologies identical with endothelium on native blood vessels. The effectiveness of endothelial cell sodding techniques in pre-clinical animal trials provides support for expanded clinical trials.     

Physiological and pathological angiogenesis in the endocrine system

Formation of new blood vessels occurs in many physiological states (during development of the embryo, cycling changes of the female reproductive tract), as well as in pathological processes (such as diabetic retinopathy and wound healing). Angiogenesis has been shown to be related to tumor formation, prognosis, and response to treatment in many tumor types. Intratumoral microvessels can be related to tumor behavior or hormone secretion in different endocrine tumors. For example, invasive prolactinomas are more vascular than noninvasive adenomas; a surgical approach is more successful in macroprolactinomas with lower microvessel density. A higher number of microvessels have been found in papillary thyroid carcinomas during recurrences. A correlation between microvessel count and prognosis in papillary and medullary thyroid carcinomas has been suggested. Several stimulating and inhibiting factors involved in the regulation of angiogenesis have been identified. Among them, vascular endothelial growth factor (VEGF) has been shown to be critically involved in angiogenesis and also in the neovascularization of solid tumors. Dopamine agonists (already in clinical use for prolactinomas) have potent inhibitory actions on VEGF signaling, and thus may be a new tool in antiangiogenic therapy. Secretion of VEGF in the great majority of human pituitary adenomas is inhibited by dexamethasone. This suggests that glucocorticoids can be considered in the treatment of certain pituitary tumors. The cyclic nature of angiogenesis in the female reproductive tract indicates that stimulation or inhibition of paracrine angiogenic factors may lead to new approaches for being able to influence reproductive endocrine disorders. Experimental and clinical aspects of interactions between angiogenic factors and tumor growth of the endocrine system are also discussed.     

Modelling of vascular growth processes: A stochastic biophysical approach to embryonic angiogenesis

In a computer simulation, growth of a capillary network is driven by a stochastic process on a planar hexagonal grid. Starting at a point source, the probabilities for the formation of new capillary elements depend on local biophysical knowledge. This knowledge is mainly derived from the flow theorem of Hagen–Poiseuille and the diameter exponent Δ. The hexagonal grid is visualized as being supported by a cylinder or a sphere. An arterial tree results from the adaptive diameter augmentation, and is considered to have limited fractal properties. The dimension of its border, and the time course of growth and of blood pressure are compared with biological data from the chorioallantoic membrane (CAM) of incubated chicken eggs. The model is discussed in view of mechanosensitivity and cell–matrix interactions of endothelial cells, and CAM haemodynamics.     

A scanning and transmission electron microscopy study of the parabronchial unit in quail (Coturnix coturnix) and town pigeons (Columba livia).

A combined scanning electron (SEM) and transmission electron microscopy (TEM) investigation was undertaken to gain insight into the complex structural pattern of the atrial compartment and the gas exchange tissue of parabronchial units in quail and town pigeons. The aim was also to depict the changes taking place in the parabronchial unit in the late prehatching and early posthatching periods in quail. The standard SEM and TEM investigation was carried out in 13 mature quail and 8 town pigeons. The developmental study involved embryonic quail (Days 15, 16, 17), newly hatched quail, quail 24 h after hatching, and quail aged 2, 10, 19, and 25 days (3 individuals per group). The luminal relief of the parabronchus is formed by anastomosing interatrial septa delineating the atrial pits, which are thinner and shallower in pigeons. The atrial bottom opens in mature individuals into 3-6 infundibula. The extracellular material represented by trilaminar substance, which does not appear until hatching, veils the surface relief of the parabronchial epithelium, which is consequently hardly accessible to three-dimensional visualization. Only in town pigeons with fewer discontinuous layers of extracellular material was it possible to visualize the surface of the atrial epithelium, that is, of the granular and squamous atrial cells. The SEM analysis has convincingly shown the intricate spatial organization of atria, infundibula, and air and blood capillaries of the gas exchange tissue. The retinacula, that is, parallelly arranged processes of squamous respiratory cells bridging the air-capillary lumina, were evidenced by SEM and TEM. The complex structure of the avian parabronchus has been successfully demonstrated in the present SEM and TEM study.     

Micro‐CT measurements of tumoral vessels supplied by portal circulation in hepatic colorectal metastasis mouse model

The purpose of this study was to elucidate the micro CT findings of tumoral vessels supplied by portal circulation during establishment of hepatic metastasis of colorectal cancer in a mouse model. Hepatic metastases were induced in 15 BALB/c mice through the injection of murine colonic adenocarcinoma tumor cells into the mesenteric vein. Micro‐CT imaging of the tumoral vessels was obtained to clarify the microvascular architecture. We evaluated the sinusoidal structure, diameter of the tumoral vessels (DTV) and blood vessel density (BVD) according to tumor sizes ranging from 201 to 3,000 µm in diameter. A total of 116 tumors were observed on day 15 after cell injection. The mean diameter of a normal hepatic sinusoid was 11.7 ± 2.0 µm on micro CT. The DTV supplied by the portal vein of tumors measuring 1,001–1,500 µm in diameter was greater than that of tumors 200–1,000 µm in diameter. The mean BVD from the portal vein gradually decrease according to size of tumor from 201 to 3,000 µm in diameter (r² = ?0.584, P < 0.01). The characteristics of tumoral vessels supplied by portal circulation during establishment of hepatic colorectal metastases were well visualized with micro‐CT imaging. Microsc. Res. Tech. 77:415–421, 2014. © 2014 Wiley Periodicals, Inc.     

Angiogenesis in endocrine glands: special reference to the expression of vascular endothelial growth factor

Recent studies have shown that several angiogenic growth factors are produced and secreted by normal endocrine cells and are increased in pathological states of endocrine glands, including inflammation, hyperplasia, and neoplasia. Expression of corresponding receptors on epithelial cells and/or endothelial cells enables these angiogenic factors to influence growth and function of the endocrine tissues by auto- or paracrine mechanisms. Some of the angiogenic factors are also considered to be involved in angiogenesis, which is a critical process in tumor formation and progression. Vascular endothelial growth factor (VEGF) is regarded as one of most important angiogenic factors with specific effects on endothelial cell growth and vascular permeability, and is isolated from a variety of normal and neoplastic endocrine cells. In this article, recent studies on angiogenic factors, especially on expression of VEGF, are reviewed in the field of endocrine systems.