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.     

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.     

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.     

Electron microscopy of cultured angiogenic endothelial cells

Angiogenesis is a multi-step process involving migration, proliferation, and a specific spatial arrangement of endothelial cells. On the basis of a model of cultured microvascular endothelial cells derived from the bovine corpus luteum, all stages of in vitro angiogenesis as well as intussusceptive remodeling were characterized by scanning and transmission electron microscopy. To preserve the delicate three-dimensional cellular structures for electron microscopy, modified processing techniques for both transmission and scanning electron microscopy including micro-corrosion casting of cultured cells were established. The detailed results on morphological alterations and cellular interactions confirmed and complemented earlier studies of in vitro angiogenesis. Electron microscopy proved to be an efficient tool for detection and supervision of all major endothelial differentiation processes resembling in vivo conditions that are generally considered important in a realistic in vitro model of angiogenesis: occurrence of function-related cellular junctions; development of specific surface features indicating cellular polarity; production of extracellular matrix material; mechanisms leading to the formation of an internal lumen; specific spatial arrangement of endothelial cells within capillary-like networks; detachment of apoptotic cells as well as intussusception of specific cells within the course of vascular remodeling. The abundance of quickly available information provided by electron microscopic approaches may be useful for subsequent, e.g., biochemical or molecular, studies and thus delivers important controls for further experimental designs.     

Cell adhesion in renal tubular epithelial cells: Biochemistry,biophysics or both

Changes in cell-cell and cell-substrate adhesion markers are increasingly used to characterize disease onset and progression. However, these relationships depend on both the biochemical and molecular association between cells and between cells and their extracellular matrix, as well as the biophysical and mechanical properties orchestrated by cytoskeletal, membrane and matrix components. To fully appreciate the role of cell adhesion when determining normal physiology and the impact of disease on cellular function, it is important to consider both biochemical and biophysical attributes of the system being investigated. In this short viewpoint we reflect on our experiences assessing cell-cell and/or cell-matrix interactions in renal tubular epithelial cells.     

Cell and matrix morpho-functional analysis in chondrocyte micromasses

Micromass cultures represent a convenient means of studying chondrocyte physiology in the context of a tridimensional culture model. In this study, we present the first ultrastructural analysis of the distribution and organization of the extracellular components in micromasses in comparison with their cartilaginous counterparts. Primary chondrocytes obtained from osteoarthritis patients were pelleted in micromasses. Transmission electron microscopy and immunofluorescence were used to evaluate the distribution of major extracellular matrix proteins, i.e., aggrecan, chondroitin-4-sulfate, chondroitin-6-sulfate, and collagen I and II. Both approaches revealed a number of morphological features shared by micromass and cartilage chondrocytes. In particular, in micromasses, chondrocytes are in close contact with an organized extracellular matrix that adequately mimics that of cartilage. Cells were observed to establish specialized junctions for cell-extracellular matrix crosstalk. Noteworthy, cells seem endowed in a chondroitin sulfate-rich microenvironment, and thus possibly ensuring the immobilization of chemokines, a family of molecules emerging in osteoarthritis pathogenesis, in a haptotactic-like gradient to the chondrocytes, which facilitates the binding to their receptors. To determine the suitability of this model to investigate osteoarthritis pathogenesis, a potential apoptotic stimulus (endothelial IL-8) was used, and ultrastructural analysis assessed apoptosis induction. Micromass cultures were proved to be an experimental technique providing a large number of properly differentiated chondrocytes, and thus allowing reliable biochemical and morphological studies. They represent, therefore, a novel approach to osteoarthritis investigation that promises more thorough understanding of chondrocyte physiology in osteoarthritis.     

Hormone regulation of endothelial apoptosis and proliferation in vessel regression and angiogenesis

Apoptosis and endothelial proliferation represent two adverse events which take place during vessel regression and angiogenesis, respectively. Apoptosis, an intrinsically activated programmed cell death, regulates cell elimination during vessel regression. In contrast, angiogenesis involves endothelial cell proliferation, migration, and vascular formation. Several molecules, including growth factors and cytokines, produced by endothelial cells and by other cells within the vicinity of the capillary network, regulate apoptosis and angiogenesis. Hormones and endocrine peptides acting via specific receptors located on the endothelial and perivascular stromal cells also have been found to be involved in the regulation of these two major antagonistic processes. The need for a better understanding of the mechanisms involved in hormone regulation of endothelial cell during apoptosis and angiogenesis is of great importance. The accumulating knowledge of hormone regulation may contribute to the introduction of new therapeutic strategies targeting the endothelial cells.     

Dancing to a somewhat different rhythm: Cell migration along the natural basement membrane

Much of our understanding of the events which underlie cell migration has been derived from studies of cells in tissue culture. One of the components that mediates this process is the dynamic actin-based microfilament system that can reorganize itself into so-called stress fibers that are considered essential components for cell motility. In contrast, relatively few studies have investigated cell movement along an extracellular matrix (ECM) which is known to influence both cellular organization and behavior. This opinion/viewpoint article briefly reviews cell migration during corneal endothelial wound repair along the tissue’s natural basement membrane, Descemet’s membrane. Because the tissue exists as a cell monolayer it affords one an opportunity to readily explore the effect of cell/matrix influences on cell motility. As such, cell movement along this substrate differs somewhat from that found in vitro and migrating endothelial cells also demonstrate an ability to move along the ECM without the benefit of having an organized actin cytoskeleton.     

Laminins: structure and genetic regulation

The laminins form a large family of modular proteins found in basement membranes, but also elsewhere. They function as structural components and are essential for morphogenesis, but in addition interact with cell surface receptors such as integrins and alpha-dystroglycan. By virtue of their receptor interactions, they initiate intracellular signalling events that regulate cellular organization and differentiation. The many interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ between different forms of laminin. In the present article, we describe how the diversity of laminins and the genetic regulation of the expression of different laminin forms lead to the formation of extracellular matrices with variable laminin composition and thereby different biological properties.     

Inhibition of angiogenesis at endothelial cell level

Targeting of proliferating endothelial cells may allow a new therapeutic strategy against malignant tumors. Endothelial cells are easily accessible through the blood stream and genetically stable, reducing the possibility of acquiring drug resistance. There are numerous candidates for angiogenesis targets of drug development, e.g. a single endothelial cell has 2,000-20,000 different receptors on a cell membrane and inside a cell there are hundreds of second messengers. Inhibitors that are active at endothelial cells can be placed in three main groups inducing blockage of endothelial cell activator, direct inhibition of endothelial cells, inhibition of endothelial specific signaling. This review summarizes different approaches already in clinical trials. Specific effort is taken to give a view of and a new perspective over clinically relevant basic mechanisms, e.g. VEGF, ETS and radionanotargeting. It is clear that aggressive development work both at the preclinical and clinical levels is still needed; however, breakthroughs in applied therapies are expected at any time.     

用于研究细胞入侵的基质降解实验

在细胞正常发育、发炎和肿瘤转移研究中有一个关键步骤,就是通过多层的细胞外基质入侵细胞.在传统的研究方法中,研究者们已经发展了自己的检测方法,就是利用荧光标记的凝胶来研究基质降解,其中伴随着细胞的入侵.这篇文章探索了研究细胞入侵的新方法,能得到比传统方法更一致的结果.研究者们利用这些方法来研究降解时间过程,扫描在降解过程中潜在的活化剂或者抑制剂,探索细胞入侵的通道.     

Role of growth factors and their receptors in gastric ulcer healing

The repair of gastric ulcers requires the reconstitution of epithelial structures and the underlying connective tissue, including vessels and muscle layers. Several growth factors have been implicated in this process, since they are able to regulate important cell functions, such as cell proliferation, migration, differentiation, secretion, and degradation of extracellular matrix, all of which are essential during tissue healing. Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), hepatocyte growth factor (HGF), and trefoil factors (TFFs) are mainly involved in the reconstitution of the epithelial structures. Platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta) play a major role in the reconstitution of connective tissue, including vessels and smooth muscle cells, and provide the extracellular matrix substrate for cell migration and differentiation. The expression of these growth factors and their receptors is increased during ulcer healing and, in some cases, intracellular signaling related to receptor binding and transduction has been demonstrated. EGF, TGF-alpha and TFFs are normally present either in the gastric juice or in the mucosa, and may exert their effects immediately after damage, before newly synthesized EGF and TFFs are released from the ulcer margin. The inhibition of their effects by neutralizing antibodies may result in delayed ulcer healing, while the administration of recombinant or natural analogues may improve ulcer repair. In this review, we will summarize the basic molecular characteristics of some of these growth factors, and will discuss available evidence supporting their role in the ulcer repair process.     

Role of MMP9 on invadopodia formation in cells from adenoid cystic carcinoma. Study by laser scanning confocal microscopy

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1‐MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent‐substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel‐gelatin‐FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1‐MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1‐MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1‐MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape

Characteristics of human malignant glioma are excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumor immune surveillance. Transforming growth factor-beta (TGF-beta), a versatile cytokine, is intimately involved in the regulation of these processes. Here, we discuss the interactions of TGF-beta with growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF), metalloproteinases (MMP-2, MMP-9) and their inhibitor, plasmin activator inhibitor-1 (PAI-1), and immune cells, like natural killer cells, T-cells and microglia. The differential effects of TGF-beta in glioma biology are outlined with emphasis on the induction of a survival advantage for glioma cells by enforced cell growth, migration, invasion, angiogenesis and immune paralysis. By virtue of its growth regulatory and immunomodulatory properties, TGF-beta promises to become a novel target for the experimental therapy of human malignant glioma.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.     

AFM studied the effect of celastrol on β1 integrin‐mediated HUVEC adhesion and migration

Integrin‐mediated human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix plays a fundamental role in tumor‐induced angiogenesis. Celastrol, a traditional Chinese medicine plant, has possessed anticancer and suppressed angiogenesis activities. Here, the mechanism underling the antiangiogenesis capacity of celastrol was investigated by exploring the effect of celastrol on β1(CD29) integrin‐mediated cell adhesion and migration. Flow cytometry results showed that the HUVECs highly expressed CD29 and cell adhesion assay indicated that celastrol specifically inhibited the adhesion of HUVECs to fibronectin (FN) without affecting nonspecific adhesion to poly‐L‐lysine (PLL). After cell FN adhesion being inhibited, the cell surface nanoscale structure and adhesion force were detected by atomic force microscope (AFM). High‐resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with celastrol. The membrane average roughness (Ra) and the major forces were decreased from 31.34 ± 4.56 nm, 519.60 ± 82.86 pN of 0 μg/ml celastrol to 18.47 ± 6.53 nm, 417.79 ± 53.35 pN of 4.0 μg/ml celastrol, 10.54 ± 2.85 nm, 258.95 ± 38.98 pN of 8.0 μg/ml celastrol, respectively. Accompanying with the decrease of adhesion force, the actin cytoskeleton in the cells was obviously disturbed by the celastrol. All of these changes influenced the migration of HUVECs from the wound‐healing migration assay. Taken together, our results suggest that celastrol can be as an inhibitor of HUVEC adhesion to FN. This work provides a novel approach to inhibition of tumor angiogenesis and tumor growth. SCANNING 35:316‐326, 2013. © 2012 Wiley Periodicals, Inc.     

Mast cells and angiogenesis

There is much evidence that angiogenesis is related to mast cells. Mast cells accumulate in many angiogenesis-dependent situations, including tumor growth, rheumatoid arthritis, ovulation, would healing, and tissue repair. Several mast cell mediators are angiogenic and regulate endothelial cell proliferation and function. Stem cell factor, vascular endothelial growth factor, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor induce chemotactic migration of mast cells to sites of neovascularization. Mast cell products such as tryptase also degrade connective tissue matrix to provide space for neovascular sprouts. Angiogenesis has been proposed as a target for anticancer therapy and for treatment of inflammatory disorders such as rheumatoid arthritis. Future studies on the cascade of angiogenic events, including mast cell-target cell interaction, and various intracellular signaling pathways are indicated to provide a new approach for the treatment of cancer and inflammatory disorders and for tissue repair.     

Protein kinases in mammary gland development and cancer

Protein kinases, the enzymes responsible for phosphorylation of a wide variety of proteins, are the largest class of genes known to regulate growth, development, and neoplastic transformation of mammary gland. Mammary gland growth and maturation consist of a series of highly ordered events involving interactions among several distinct cell types that are regulated by complex interactions among many steroid hormones and growth factors. The mammary gland is one of the few organ systems in mammals that complete their morphologic development postnatally during two discrete physiologic states, puberty and pregnancy. Thus, the mammary gland is an excellent model for studying normal development and the early steps of tumor formation. The susceptibility of the mammary gland to tumorigenesis is influenced by its normal development, particularly during stages of puberty and pregnancy. Numerous experimental and epidemiological studies have suggested that specific details in the development of the mammary gland play a critical role in breast cancer risk. Mammary gland development is characterized by dynamic changes in the expression and functions of protein kinases. Perturbations in the regulated expression or function of protein kinases or their associated signaling pathways can lead to malignant transformation of the breast. For example, overexpression of several receptor-tyrosine kinases, including human epidermal growth factor receptor and HER2/Neu, has been shown to contribute to the development of breast cancer. Since receptor-tyrosine kinases regulate several essential processes such as mitogenesis, motility, invasion, cell survival, and angiogenesis, targeting receptor-tyrosine kinases may have important implications in designing strategies against breast cancer.     

Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations of specific collagen fragments believed to discriminate between cathepsin K and MMP cleavage. Next, we provide evidence that MMPs are very critical for osteoclast migration, thereby controlling also the cell-matrix interactions required for cell attachment/detachment. The evidence supporting this role is based on a model of osteoclast recruitment in primitive long bones, an assay of osteoclast invasion through collagen gel, and the effect of proteinase inhibitors/knockouts in these models. Furthermore, we mention observations indicating a role of MMPs in initiation of bone resorption. Finally, we emphasize the many distinct ways MMPs may alter focally the extracellular environment thereby regulating the osteoclast behavior. Although the understanding of MMPs in osteoclast biology is rapidly expanding, it is suspected that important roles remain to be discovered.     

Cell-substrate interactions in cnidaria

Studies on morphogenesis and regeneration in cnidarians have a long history, and the importance of cell-ECM (extracellular matrix) interactions for these processes has been well recognized and studied since the middle of the 20th century. Cnidarians have a life cycle with a larva, a polyp, and often a medusa generation. In the medusa, the ECM (mesoglea) is very prominent and essentially shapes the animal. In the larva and the polyp, the ECM is a thin layer. Some of the ECM components known from vertebrates have been identified in cnidarians by immunohistochemistry, electron microscopy, rotary shadowing, biochemistry, and molecular cloning. In vivo and in vitro experiments suggest that the cnidarian ECM plays a role in cell migration and morphogenesis comparable to that known from other developmental systems. In the fresh water polyp Hydra, regeneration of body patterns and migration of nematocytes seems to require the presence of ECM ligands and the corresponding cell receptors. In hydrozoan medusae, DNA replication and the stability of the differentiated state of isolated tissue can be influenced by altering the properties of the ECM substrate. When cultured, most cnidarian cells survive only when attached to ECM substrates, they rarely divide and die within short times.