Pathogenic angiogenesis in IBD and experimental colitis: new ideas and therapeutic avenues

Angiogenesis is now understood to play a major role in the pathology of chronic inflammatory diseases and is indicated to exacerbate disease pathology. Recent evidence shows that angiogenesis is crucial during inflammatory bowel disease (IBD) and in experimental models of colitis. Examination of the relationship between angiogenesis and inflammation in experimental colitis shows that initiating factors for these responses simultaneously increase as disease progresses and correlate in magnitude. Recent studies show that inhibition of the inflammatory response attenuates angiogenesis to a similar degree and, importantly, that inhibition of angiogenesis does the same to inflammation. Recent data provide evidence that differential regulation of the angiogenic mediators involved in IBD-associated chronic inflammation is the root of this pathological angiogenesis. Many factors are involved in this phenomenon, including growth factors/cytokines, chemokines, adhesion molecules, integrins, matrix-associated molecules, and signaling targets. These factors are produced by various vascular, inflammatory, and immune cell types that are involved in IBD pathology. Moreover, recent studies provide evidence that antiangiogenic therapy is a novel and effective approach for IBD treatment. Here we review the role of pathological angiogenesis during IBD and experimental colitis and discuss the therapeutic avenues this recent knowledge has revealed.     

Cutting edge: proangiogenic properties of alternatively activated dendritic cells

Angiogenesis plays an important role in tissue remodeling and repair during the late phase of inflammation. In the present study, we show that human dendritic cells (DC) that matured in the presence of anti-inflammatory molecules such as calcitriol, PGE2, or IL-10 (alternatively activated DC) selectively secrete the potent angiogenic cytokine vascular endothelial growth factor (VEGF) isoforms VEGF165 and VEGF121. No VEGF production was observed in immature or classically activated DC. Also, the capacity to produce VEGF was restricted to the myeloid DC subset. When implanted in the chick embryo chorioallantoic membrane, alternatively activated DC elicit a marked angiogenic response, which is inhibited by neutralizing anti-VEGF Abs and by the VEGFR-2 inhibitor SU5416. Therefore, alternatively activated DC may contribute to the resolution of the inflammatory reaction by promoting VEGF-induced angiogenesis.     

Angiogenesis in hypersensitivity pneumonitis

Angiogenesis is an essential process required for growth and tissue repair after injury, but it may also contribute to the pathology of a number of human disorders including neoplasias, atherosclerosis and inflammatory diseases. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide upregulated by many cytokines and endothelium shear stresses. Lung is a highly vascular tissue with finely organized and regulated microvascular beds, and its inflammation may lead to dysregulated angiogenesis. Hypersensitivity pneumonitis (HP) is a lung disorder characterized by chronic lymphocytic inflammation and endothelial damage. However, neovascularization has not been previously explored. In this study, we examined the expression and localization of VEGF in 38 patients with HP and 14 healthy control subjects (CS). VEGF levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA, and cellular lung localization was determined by immunohistochemistry. In addition, VEGF expression was analyzed in lung tissue by RT-PCR. Our results showed sera levels significantly increased in HP patients compared with CS (209.3 +/- 189.3 vs. 55.3 +/- 31.4 pg/ml; p = 0.004). By contrast, BALF levels of VEGF were significantly decreased in HP patients compared with CS (35.3 +/- 51.5 pg/ml vs. 185.1 +/- 191.4 pg/ml; p < 0.001). VEGF was primary expressed by epithelial cells, smooth muscle cells, and interstitial macrophages in HP tissue. Flt-1 and Flk-1 receptors were highly expressed by endothelial cells from medium and small vessels in HP tissue. By RT-PCR the VEGF RNA was increased compared with those in normal lung. Our results suggest that abnormal expression of VEGF may contribute to impair the lung repair in HP.     

Vascular endothelial growth factor (VEGF) - part 1: in physiology and pathophysiology

Angiogenesis is an important component of many physiological processes, such as the female sexual cycle, placenta formation, the processes of growth and differentiation of tissues, and reparative processes including wound healing, fracture repair, and liver regeneration. The formation of new blood vessels during angiogenesis and vasculogenesis allows the growth and functioning of multicellular organisms. Pathological angiogenesis most commonly occurs in ischaemic, inflammatory and neoplastic diseases. Conditions in the pathogenesis of which angiogenesis plays an important role are sometimes labelled angiogenic diseases. To date, a number of pro-and anti-angiogenic factors have been defined. VEGF is the only specific mitogen for endothelial cells. It stimulates their growth and inhibits apoptosis, increases vascular permeability in many tissues, promotes vasculogenesis and angiogenesis. VEGF signalling activity in relation to the cell is dependent on having its specific membrane receptors (Flt-1, KDR, Flt-4). Angiogenesis plays a protective role in ischaemic heart disease and myocardial infarction. Angiogenesis extends life for patients after a stroke. Most of the facts about physiological angiogenesis are derived from studies into liver regeneration as a result of an acute injury or partial hepatectomy. Pathological hepatic angiogenesis occurs in the course of inflammation, fibrosis, hypoxia, and during tumourogenesis. There is interesting data relating to liver steatosis and obesity.     

Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat

Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.     

Angiogenesis and chemokines in rheumatoid arthritis and other systemic inflammatory rheumatic diseases

Angiogenesis, the formation of new vessels, is important in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. Chemotactic cytokines termed chemokines mediate the ingress of leukocytes, including neutrophils and monocytes into the inflamed synovium. In this review, authors discuss the role of the most important angiogenic factors and angiogenesis inhibitors, as well as relevant chemokines and chemokine receptors involved in chronic inflammatory rheumatic diseases. RA was chosen as a prototype to discuss these issues, as the majority of studies on the role of angiogenesis and chemokines in inflammatory diseases were carried out in arthritis. However, other systemic inflammatory (autoimmune) diseases including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SS), mixed connective tissue disease (MCTD), polymyositis/dermatomyositis (PM/DM) and systemic vasculites are also discussed in this context. As a number of chemokines may also play a role in neovascularizaton, this issue is also described here. Apart from discussing the pathogenic role of angiogenesis and chemokines, authors also review the regulation of angiogenesis and chemokine production by other inflammatory meditors, as well as the important relevance of neovascularization and chemokines for antirheumatic intervention.     

Chemokine Localization in Bronchial Angiogenesis

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0–24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.     

An intracrine view of angiogenesis

Angiogenesis, the generation of new blood vessels from pre-existing vessels, is an integral component of wound healing, responses to inflammation and other physiologic processes. It is also an essential part of tumor growth; in the absence of new vessel formation, tumors cannot expand beyond a small volume. Although much is known about angiogenesis and its regulation, there is no overall theory that describes or explains this process. It is here suggested that the intracrine hypothesis, which ascribes to certain extracellular signaling peptides (whether hormones, growth factors, DNA-binding proteins or enzymes) a role in both intracellular biology and extracellular signaling, can contribute to a more general understanding of angiogenesis. Intracrine factors participate in angiogenesis in the following ways: (1) they can act within the cells that synthesized them (type I intracrine action), (2) they can be secreted and then taken up by their cell of synthesis to act intracellularly (type II intracrine action ), or (3) they can be secreted and internalized by a distant target cell (type III intracrine action). The parallels between the intracrine growth factor mechanisms cancer cells employ in stimulating their own growth and the mechanisms operative in endothelial cell proliferation during angiogenesis ("intracrine reciprocity") are discussed. Collectively, these explorations lead to testable hypotheses regarding the regulation of normal and pathological angiogenesis, and point to similarities between tumor-induced angiogenesis and tissue differentiation.     

Regulation of angiogenesis-expression of VEGF receptors]

Angiogenesis, the formation of new blood vessels from pre-existing endothelium, is a crucial process for tumor growth, metastasis and inflammation. Therefore, it is focused on the anti-tumor therapy to prohibit angiogenesis in animal model and clinical studies. Eicosapentaenoic acid (EPA 20: 5,n-3) can restrain tumor growth and inflammation. In this paper, we examined the effects of EPA on tube formation. In EPA-pretreated endothelial cells angiogenesis was attenuated and also proliferation induced by VEGF, but not by b-FGF, was suppressed. The reason why EPA suppressed endothelial cell proliferation induced by VEGF was that EPA selectively inhibited the expression of KDR. As we mentioned, the regulation of angiogenesis in vivo may be involved in the expression of VEGF receptors.     

The resolution of inflammation and cancer

Inflammation has long been thought to contribute to the development of cancer; however there is also clear evidence that the immune system can recognize and eliminate cancer cells. Current research suggests that cancer-associated inflammation has a dual role in tumor progression; inflammatory mediators promote the malignant activity of cancer cells by acting as growth factors and also stimulate angiogenesis, however, cancer-associated inflammation is also linked with immune-suppression that allows cancer cells to evade detection by the immune system. In this review we will discuss the dual role of inflammation in cancer and how endogenous anti-inflammatory mechanisms may equally be important in carcinogenesis.     

Angiogenesis and organ transplantation

Angiogenesis is a vessel development process that maintains the vascular supply for organ function. Regulation of angiogenesis is provided by positive factors, such as vascular endothelial or basic fibroblast growth factors, and negative factors, such as thrombospondin and macrophage-derived inflammatory cytokines. While the role of angiogenesis in the wound healing, embryogenesis, tumor growth and proliferative diseases is clear, in organ transplantation it is not yet well established. Herein we discuss the potential role of angiogenesis in chronic renal disease and in transplant settings.     

Relationship between angiogenesis and inflammation in experimental arthritis

Background. Angiogenesis is involved in rheumatoid arthritis (RA) leading to leucocyte recruitment and inflammation in the synovium. Furthermore, synovial inflammation itself further potentiates endothelial proliferation and angiogenesis. In this study, we aimed at evaluating the reciprocical relationship between synovial inflammation and angiogenesis in a RA model, namely collagen-induced arthritis (CIA). Methods. CIA was induced by immunization of DBA/1 mice with collagen type II in adjuvant. Endothelial cells were detected using a GSL-1 lectin-specific immunohistochemical staining on knee joint sections. Angiogenesis, clinical scores and histological signs of arthritis were evaluated from the induction of CIA until the end of the experiment. Angiogenesis was quantified by counting both the isolated endothelial cells and vessels stained on each section. To evaluate the effect of increased angiogenesis on CIA, VEGF gene transfer was performed using an adeno-associated virus encoding VEGF (AAV-VEGF), by intra-muscular or intra-articular injection in mice with CIA. Results. We showed an increase in synovial angiogenesis from day 6 to day 55 after CIA induction, and, moreover, joint vascularization and clinical scores of arthritis were correlated (p < 0.0001, r = 0.61). Vascularization and histological scores were also correlated (p = 0.0006, r = 0.51). Systemic VEGF overexpression in mice with CIA was followed by an aggravation of arthritis as compared to AAV-lacZ control group (p < 0.0001). In contrast, there was no difference in clinical scores between control mice and mice injected within the knee with AAV-VEGF, even if joint vascularization was higher in this group than in all other groups (p = 0,05 versus non-injected group). Intra-articular AAV-VEGF injections induced more severe signs of histological inflammation and bone destruction than AAV-Lac Z or no injection. Conclusion. Angiogenesis and joint inflammation evolve in parallel during collagen-induced arthritis. Furthermore, this work shows that exogenous VEGF can aggravate CIA. It is direct evidence that the increase in joint vascularization leads to an exacerbation of arthritis. Taken together, these results emphasize the role of angiogenesis in inflammatory arthritis. It also suggests an early involvement of angiogenesis in joint inflammation.     

Cigarette smoke extract inhibits angiogenesis of pulmonary artery endothelial cells: the role of calpain

Angiogenesis is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the lung tissue remodeling associated with cigarette smoke-induced chronic obstructive pulmonary disease (COPD). To investigate the role of angiogenesis in the pathogenesis of COPD, we evaluated the effect of cigarette smoke extract (CSE) on angiogenesis of pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 2.5-10% CSE resulted in a dose-dependent inhibition of endothelial monolayer wound repair. CSE also caused inhibition of tube formation on Matrigel, migration in a Boyden chamber, and proliferation of PAEC. Because calpain, a family of calcium-dependent intracellular proteases, mediates cytoskeletal signaling in endothelial motility, we explored the role of calpain in the CSE-induced inhibition of endothelial angiogenesis. Incubation of CSE resulted in a dose-dependent decrease in calpain activity. Calpain inhibitor-1, a specific inhibitor of calpain, potentiates inhibitory effect of CSE on the endothelial monolayer wound repair, tube formation, cell migration, and cell proliferation. Transfection of PAEC with antisense oligodeoxyribonucleotides of calpastatin, the major endogenous calpain inhibitor, prevented CSE-induced increase in calpastatin protein content and CSE-induced decreases in calpain activity. It also prevented CSE-induced decreases in monolayer wound repair, tube formation, and migration. These results suggest that CSE attenuates angiogenesis of PAEC and the mechanism involves inhibition of calpain. Impaired angiogenesis may impede the repair process in the lungs of cigarette smokers and contribute to the altered structural remodeling observed in the lungs of patients with cigarette smoke-related COPD.     

Nitric oxide signaling during myocardial angiogenesis

Ischemic heart disease develops as a consequence of coronary atherosclerotic lesion formation. Coronary collateral vessels and microvascular angiogenesis develop as an adaptive response to myocardial ischemia, which ameliorates the function of the damaged heart. Angiogenesis, the formation of new blood vessels from pre-existing vascular bed, is of paramount importance in the maintenance of vascular integrity both in the repair process of damaged tissue and in the formation of collateral vessels in response to tissue ischemia. Angiogenesis is modulated by a multitude of cytokines/chemokines and growth factors. In this regard, angiogenesis cannot be viewed as a single process. It is likely that different mediators are involved in different phases of angiogenesis. Vascular endothelial cells (ECs) produce nitric oxide (NO), an endothelium-derived labile molecule, which maintains vascular homeostasis and thereby prevents vascular atherosclerotic changes. In patients with ischemic heart disease, the release of endothelium-derived NO is decreased, which plays an important role in the atherosclerotic disease progression. In recent years, endothelium-derived NO has been shown to modulate angiogenesis in vitro and in vivo. In this review, we summarize recent progress in the field of the NO-mediated regulation of postnatal angiogenesis, particularly in response to myocardial ischemia.     

Adipose tissue remodeling and chronic inflammation in obesity visualized by in vivo molecular imaging method

Obese visceral adipose tissue remodeling and dysfunction, based on chronic inflammation and local immunological changes, play major roles in the metabolic syndrome. Therefore, an in vivo visualization technique has been developed to assess the dynamic interplay between multiple cell types in obese adipose. In vivo imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis, which were augmented in obese adipose tissue. In addition, increased leukocyte–platelet–endothelial cell interactions were observed in the microcirculation, a hallmark of inflammation. Upregulated expression of adhesion molecules contribute to the local activation of inflammatory processes. We also found that large numbers of CD8+ effector T cells infiltrated into the obese adipose tissue, playing major roles in inflammatory macrophage infiltration into obese adipose tissue, the induction and maintenance of inflammation, and systemic insulin resistance. Our results demonstrate the power of our imaging technique to analyze multi-cellular interactions in inflammation in vivo and to evaluate new therapeutic interventions.     

Proangiogenic function of CD40 ligand-CD40 interactions

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.     

Alteration in the endothelial cell functional activity by the <Emphasis Type="Italic">Streptoccocus pyogenes</Emphasis> ultrasonic lysate

Angiogenesis and vascular remodeling are vital inflammation components. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. Here, we examined angiogenic properties of EA.hy 926 human endothelial cells treated with ultrasonic lysate of Streptoccocus pyogenes. It was found that the lysate inhibited the cell adhesion, migration, metabolism and proliferation in a dose-dependent manner but even in the highest dose it did not affect the cells viability. Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. Taking together, our results suggest that impaired angiogenic function of endothelial cells may contribute to the tissue perfusion reduction, hypoxia development and subsequent regional tissue necrosis caused by Streptococci group A.     

Impact of modeled microgravity on microvascular endothelial cells

Microvascular endothelial cells are protagonists in inflammation and angiogenesis. They contribute to the integrity of microvasculature by synthesizing a large array of cytokines, growth factors and mediators active on the endothelium itself, on smooth muscle cells and circulating leukocytes. Because space flight (i) associates with vascular impairment and (ii) modulates the cytokine network, we evaluated the effect of modeled microgravity on microvascular 1G11 cells. We found that modeled microgravity reversibly inhibits endothelial growth and this correlates with an upregulation of p21, a cyclin-dependent kinases inhibitor. By protein array, we found that microgravity inhibits the synthesis of interleukin 6, an event that may contribute to growth retardation. We also detected increased amounts of nitric oxide, a mediator of inflammatory responses, a potent vasodilator and a player in angiogenesis. The increased synthesis of nitric oxide is due, at least in part, to an upregulation of endothelial nitric oxide synthase. Because low levels of IL-6 might contribute to endothelial growth retardation as well as to the enhancement of nitric oxide synthesis, we hypothesize a central role of IL-6 in modulating microvascular endothelial cell behaviour in modeled microgravity.