Angiogenesis in breast cancer: the role of transforming growth factor beta and CD105

The progression of breast cancer depends on the establishment of a neovasculature, by a process called angiogenesis. Angiogenesis is an invasive cellular event that requires the co-ordination of numerous molecules including growth factors and their receptors, extracellular proteins, adhesion molecules, and proteolytic enzymes. TGFbeta has emerged to be a major modulator of angiogenesis by regulating endothelial cell proliferation, migration, extracellular matrix (ECM) metabolism, and the expression of adhesion molecules. It is a potent growth inhibitor of normal mammary epithelial cells and a number of breast cancer cell lines. It seems that TGFbeta exerts pleiotropic effects in the oncogenesis of breast cancers in a contextual manner, i.e., it suppresses tumourigenesis at an early stage by direct inhibition of angiogenesis and tumour cell growth. However, over-production of TGFbeta by an advanced tumour may accelerate disease progression through indirect stimulation of angiogenesis and immune suppression. The cell membrane antigen CD105 (endoglin) binds TGFbeta1 and TGFbeta3 and is preferentially expressed in angiogenic vascular endothelial cells. The reduction of CD105 levels in HUVEC leads to in vitro angiogenesis inhibition and massive cell mortality in the presence of TGFbeta1. CD105 null mice die in utero with impaired vasculature, indicating the pivotal role of CD105 in vascular development. The administration of an immunotoxin-conjugate, mab to CD105, induces long-term and complete regression of breast cancer growth in SCID mice. Therefore, CD105 is a promising vascular target for antiangiogenic therapy.     

Structure and function of the extracellular matrix of anuran eggs

The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure-function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm-triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laevis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin-ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin-ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM.     

Bone repair investigation using rhBMP-2 and angiogenic protein extracted from latex

Background: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP‐2) and P‐1 application, using 5 and 10 μg of each, combined to a material carrier, in critical bone defects. Methods: It was used 70 Wistar rats (male, ～250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 μg of pure P‐1, 5 μg of pure rhBMP‐2, 5 μg of P‐1/monoolein gel, 5 μg of rhBMP‐2/monoolein gel, 10 μg of pure P‐1, 10 μg of pure rhBMP‐2, 10 μg of P‐1/monoolein gel, 10 μg of rhBMP‐2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. Results: Animals treated with pure P‐1 protein, in both situations with 5 μg and 10 μg, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 μg were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 μg rhBMP‐2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. Conclusion: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP‐2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent. Microsc. Res. Tech., 2011.© 2011 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.     

Fertilization-Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self-assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotaryshadowed eggs, are illustrated.     

Light and low-frequency pulsatile hydrostatic pressure enhances extracellular matrix formation by bone marrow mesenchymal cells: an in-vitro study with special reference to cartilage repair

Ovine bone marrow mesenchymal cells (BMMCs) were seeded on to non-woven filamentous plasma-treated polyester scaffolds and cultured in a chondrogenic medium for 4 weeks. Thereafter a pulsatile hydrostatic pressure (PHP) was applied to these cell-scaffolds constructs at an amplitude of 0.1 MPa and frequency of 0.25 Hz, for 30 min a day, over a period of 10 days. Samples (n = 6) were removed 24 h after PHP stimulation at days 1, 4, 7, and 10 for biochemical analysis. Similar analyses were conducted, at the same time points, on control samples that were not subjected to a PHP. The results showed that the glycosaminoglycan (GAG) content did not significantly increase until after the application of a PHP for 7 days. The GAG content was 1.5 and 2.7 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The deoxyribonucleic acid (DNA) content was 1.5 times greater in the PHP group than in the control group at day 10 (p<0.01). GAG synthesis amounts, expressed as the total GAG contents per microgram of DNA, were 1.6 and 1.8 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The total collagen content in the medium did not change until after PHP application for 10 days, when it was 1.9 times higher than the control (p < 0.05). The results suggest that a light PHP applied at a low frequency has a cumulative stimulatory effect on the BMMCs' metabolic activities including cell proliferation and synthesis of the extracellular matrix.     

Preservation and visualization of the sea urchin embryo blastocoelic extracellular matrix.

Several methods were utilized to visualize the structure and orientation of the blastocoelic extracellular matrix (ECM) in Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Rapid freezing in liquid propane cooled to LN2 temperatures followed by freeze substitution was used to preserve the ECM without shrinkage due to dehydration. Scanning, transmission, and light microscopy were employed to elucidate the ECMs' structure. The blastocoelic ECM consisted of parallel fibrillar sheets that were interconnected by finer filaments and oriented along the animal-vegetal axis. The ECM completely filled the blastocoelic cavity as viewed by scanning electron microscopy. The basal lamina could be distinguished from the blastocoelic ECM as a thin coat on the plasma membrane of epithelial cells; the ECM was in contact with this coat. In contrast, the blastocoelic ECM attached directly to the plasma membrane of primary mesenchyme cells (PMC) which did not possess a basal lamina. The blastocoelic ECM was isolated as an intact "bag" and probed in a hydrated state with Con A and alcian blue. Confocal microscopy confirmed that the entire blastocoel was filled with a fibrillar ECM. These approaches offer advantages for future studies of the ECMs of sea urchin embryos and their roles in gastrulation.     

Microvascular growth, development, and remodeling in the embryonic avian kidney: the interplay between sprouting and intussusceptive angiogenic mechanisms

Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.     

Deployment of extracellular matrix proteins in sea urchin embryogenesis.

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi-laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle.     

Mechanotransduction-The relationship between gravity,cells and tensile loading in extracellular matrix

Gravity plays a central role in vertebrate development and evolution. Mechanotransduction involves the tensile tethering of veins and arteries, connections between the epidermis and dermis in skin, tensile stress concentrations that occur at tissue interfaces, cell-cell interactions, cell-collagen fiber stress transfer in extracellular matrix and fluid shear flow. While attention in the past has been directed at understanding the myriad of biochemical players associated with mechanotransduction pathways, less attention has been focused on determining the tensile mechanical behavior of tissues in vivo. Fibroblasts sit on the surface of collagen fibers in living skin and exert a retractile force on the fibers. This retractile force pulls against the tension in collagen fibers in skin. After fibroblast-collagen fiber interactions are altered either by changes in fibroblast adhesion or after formation of cancer associated fibroblasts, and changes in cell junctions, alterations in the retractive force leads to changes in mechanotransduction. The purpose of this paper is to present a model of tensile forces that occur at the fibroblast-collagen fiber interface and how these forces are important in extracellular matrix physiology in health and disease.     

Possible roles of extracellular matrix and cytoskeleton in leech body wall muscles

Round circomyarian fibres of leeches are peculiar helical muscles. The fibres are characterized by a lack of junctions, being separated by a thick extracellular matrix, and by scarce end-plates. Even so, the fibres grouped in units show the same degree of contraction. Biochemical, immunocytochemical and ultrastructural studies were performed in order: (a) to demonstrate the presence in the extracellular matrix of fibronectin, collagen type IV and laminin and in the cytoskeleton of desmin and α-actinin; (b) to show the possible link of extracellular matrix with the scaffold of intermediate filaments; (c) to evaluate how the extracellular matrix can play a role in the transduction of a signal during contraction–relaxation–superelongation phases.     

A method for TEM visualization of the extracellular matrix three-dimensional organization in tissues

A method for obtaining high-resolution three-dimensional images of the extracellular matrix organization in tissues is described. It consists of TEM observation of rotary-shadowed platinum–carbon replicas obtained from critical-point dried resinless sections of polyethylene glycol-embedded specimens. The procedure is simple and rapid, with high rates of sample recovery. An example of its application to EM immunocytochemistry (fibronectin localization) is presented. The utilization of the method to demonstrate cell-extracellular matrix relationships, and its limitations in the study of cells are discussed.     

Microscopical evaluation of extracellular matrix and its relation to the palatopharyngeal muscle in obstructive sleep apnea

Obstructive sleep apnea hypopnea syndrome (SAHS) is a complex disease of the upper respiratory airways. SAHS physiopathology is multifactorial in which airway compliance is a very important component. To evaluate the tissue changes in the palatopharyngeal muscle by morphometric, histochemical, immunohistochemical, and stereological quantification, with special attention to extracellular matrix associated with this muscle at the structural and ultrastructural levels. Thirty patients with SAHS were divided into groups of 10 according to disease severity: mild, moderate, and severe SAHS. In addition, the control group consisted of 10 patients. Fragments of palatopharyngeal muscle removed from patients with SAHS and tonsillectomies from patients in the control group were histopathologically submitted to light microscopy and transmission electron microscopy. Histopathological evaluations by light and transmission electron microscopes showed differences in analyzed groups, such as reduction of the muscle fiber diameter in patients with SAHS, taking disease severity into consideration. In contrast, stereological analysis showed a gradual increase of the collagen and elastic system fibers relative frequencies, proportionally to SAHS seriousness. MMP‐2 and MMP‐9 immunostaining also showed an increased reaction in the muscle fiber cytoplasm and endomisium during SAHS progression. The ultrastructural analysis showed that palatopharyngeal muscle fibers presented cytoplasmic residual corpuscles, a sign of early cell aging. In conclusion, the increase of tissue compliance in individuals with SAHS can be, in addition to other factors, consequence of diminished contractile activity of the muscle fibers, which exhibited clear signs of early senescence. Moreover, extracellular matrix components changes may contribute to muscle myopathy during SAHS progression. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Biochemical and morphological alterations of the extracellular matrix of chicken calcaneal tendon during maturation

The region in tendons that surrounds bone extremities adapts to compression forces, developing a fibrocartilaginous structure. During maturation, changes occur in the amount and organization of macromolecules of the extracellular matrix of tendons, changing the tissue morphology. To study the effect of maturation on tendons, Pedrês chickens were sacrificed at 1, 5, and 8 months old and had the calcaneal tendon (CT) divided into proximal region, submitted to tension/compression forces ( p ), and distal region submitted to tension force ( d ). Morphological analysis of the p region showed the presence of fibrocartilage in all ages. In the central part of the fibrocartilage, near a diminishment of the metachromasy, there was also a development of a probable fat pad that increased with the maturation. The activity of MMP‐2 and MMP‐9 was higher at 5 and 8 months old, in both regions, compared with 1‐month‐old animals. In SDS‐PAGE analysis, components with electrophoretic migration similar to decorin and fibromodulin increased with maturation, particularly in the d region. The Western blotting confirmed the increased amount of fibromodulin with maturation. In conclusion, our results show that process of maturation leads to the appearance of a probable fat pad in the center of the fibrocartilage of CT, with a reduced amount of glycosaminoglycans and an increased activity of MMPs. Microsc. Res. Tech. 78:949–957, 2015. © 2015 Wiley Periodicals, Inc.     

Angiogenesis in cutaneous melanoma: pathogenesis and clinical implications

Neovacularization is an essential step in the multistage progression of malignant melanoma. The onset of new blood vessel formation is ushered in by the release of VEGF and numerous other angiogenic molecules by the tumor cells. Human melanoma is unique among neoplasms that both avascular (early horizontal growth phase characterized by very slow progression and 99%, 10-year survival) and vascular (late radial and vertical growth phase associated with rapid growth, metastasis and death in many cases), phases are discernible by the naked eye. Although cell biologists have made great strides in unraveling the mechanisms involved in the laying down of tumor vasculature and the factors that inhibit it, clinicians treating melanoma have been rather slow to realize and utilize the full potential of suppressing the tumor blood flow to the best advantage of the patient. We suggest a consorted endeavor by all the melanoma experts across the globe to establish an "angiogenesis database" wherein they pool the blood flow and vascularity information along with Breslow's thickness, Clark's level of invasion, lymphatic and vascular invasion, regression, and outcome of their patients.     

Early mineralization of matrix vesicles in the epiphyseal growth plate

Matrix vesicles (MVs) induce the primary mineralization in collagen-rich hard tissues such as bone, mineralizing cartilage and dentine. Calcium and phosphate ions accumulate at the inner MV membrane. This accumulation takes place in association with phospholipids alone and/or in association with Annexin V, which displays Ca ion channel activity when inserted in membranes; consequently, Annexin V may be involved in Ca uptake by matrix vesicles. The first crystal nuclei are formed at these macromolecules of the MV inner membrane. They grow to stable nanometre-sized particles, dots, which coalesce to form chains of dots along the macromolecules of the MV inner membrane. At the same time, or shortly afterwards, chains of these Ca phosphate dots also develop inside the MVs. The measured centre-to-centre distances between these dots represent approximately the distances between the nucleating sites, called active sites, along the MV matrix molecules. The mineralization does not stop at the MV membrane but expands continuously into the extravesicular region in radial directions to form nodules. These radiating Ca phosphate chains, which coalesce to form needles, are composed of such primary dots, which have developed at the nucleating sites of the corresponding macromolecules.     

肛周脓肿肛瘘术后创面愈合影响因素分析

目的:分析肛周脓肿肛瘘术后创面愈合影响因素,为患者术后创面愈合质量的改善提供参考依据。方法:研究对象为2014年5月~2016年5月于我科行手术治疗的175例肛周脓肿、肛瘘患者。根据患者术后7 d创面分泌物、肉芽组织生长、创面疼痛情况,评价其创面愈合质量,并比较不同基线资料患者创面愈合质量的差异,运用多因素Logistic回归分析,总结影响肛周脓肿肛瘘术后创面愈合的危险因素。结果:术后便秘或便秘史、营养饮食不合理、机械刺激、术后感染、合并慢性全身性消耗疾病是影响肛周脓肿、肛瘘术后创面愈合质量的独立危险因素(P<0.05)。结论:肛周脓肿、肛瘘术后创面愈合质量受患者自身状态、术后处理等多种因素影响,针对上述危险因素,采取积极有效的措施加以干预有望改善创面愈合质量,保证患者术后顺利康复。     

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.     

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.