Tryptase and chymase are angiogenic in vivo in the chorioallantoic membrane assay

Human mast cells (MCs) are divided in two types depending on the expression of tryptase and chymase in their granules. Literature data indicate that both tryptase and chymase are angiogenic, but there is currently no evidence of their direct angiogenic activity in vivo. In this study, we have investigated the capacity of tryptase and chymase to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay to study angiogenesis and anti-angiogenesis. The results showed that both tryptase and chymase stimulate angiogenesis and that the response is similar to that obtained with vascular endothelial growth factor (VEGF), a well-known angiogenic cytokine, and confirm the angiogenic activity of these two proteases stored in MC granules.     

Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP‐Dependent Mechanism and Wound Angiogenesis

Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar‐free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta‐adrenoceptors (β‐AR) are G protein‐coupled receptors (GPCRs) expressed on all skin cell‐types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β‐AR‐mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β‐AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)‐dependent and protein kinase A (PKA)‐independent mechanisms as demonstrated through use of an EPAC agonist that auto‐inhibited the cAMP‐mediated β‐AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β‐AR activation reduced pro‐angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β‐AR‐mediated autocrine and paracrine anti‐angiogenic mechanisms. In more complex environments, β‐AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β‐AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β‐AR agonists could be promising anti‐angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Chronic morphine treatment inhibits LPS-induced angiogenesis: Implications in wound healing

Delayed wound healing is a chronic problem in opioid drug abusers. We investigated the role chronic morphine plays on later stages of wound healing events using an angiogenesis model. Our results show that morphine treatment resulted in a significant decrease in inflammation induced angiogenesis. To delineate the mechanisms involved we investigate the role of hypoxia inducible factor 1 alpha (HIF-1 alpha), a potent inducer of angiogenic growth factor. Morphine treatment resulted in a significant decrease in the expression and nuclear translocation of HIF-1 alpha with a concurrent suppression in vascular endothelial growth factor (VEGF) synthesis. Cells of the innate immune system play a dominant role in the angiogenic process. Morphine treatment inhibited early recruitment of both neutrophils and monocytes towards an inflammatory signal with a significant decrease in the monocyte chemoattractant MCP-1. Taken together, our studies show that morphine regulates the wound repair process on multiple levels. Morphine acts both directly and indirectly in suppressing angiogenesis.     

Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.     

Carbon inhibits vascular endothelial growth factor- and fibroblast growth factor-promoted angiogenesis

Angiogenesis is important for normal growth and wound healing processes. An imbalance of the growth factors involved in this process, however, causes the acceleration of several diseases including malignant, ocular, and inflammatory diseases. Inhibiting angiogenesis through interfering with its pathway is a promising methodology to hinder the progression of these diseases. Herein, we studied the anti-angiogenic effects of various carbon materials such as graphite, multiwalled carbon nanotubes and fullerenes in vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2)-induced angiogenesis evaluated in the chick chorioallantoic membrane (CAM) model. All the carbon materials tested showed substantial anti-angiogenic activity against either FGF2- or VEGF-induced angiogenesis in the CAM model. Those carbon materials did not have any significant effects on basal angiogenesis in the absence of the added growth factors.     

Effects of nerve growth factor (NGF) on blood vessels area and expression of the angiogenic factors VEGF and TGFbeta1 in the rat ovary

Background  

Angiogenesis is a crucial process in follicular development and luteogenesis. The nerve growth factor (NGF) promotes angiogenesis in various tissues. An impaired production of this neurotrophin has been associated with delayed wound healing. A variety of ovarian functions are regulated by NGF, but its effects on ovarian angiogenesis remain unknown. The aim of this study was to elucidate if NGF modulates 1) the amount of follicular blood vessels and 2) ovarian expression of two angiogenic factors: vascular endothelial growth factor (VEGF) and transforming growth factor beta 1 (TGFbeta1), in the rat ovary.     

Osteocalcin is angiogenic in vivo

The exact function of osteocalcin (OC), a protein synthesized by osteoblasts during the matrix mineralization phase, is still unknown. In this study we investigated the capacity of OC to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay for angiogenesis and anti-angiogenesis. The results showed that OC stimulates angiogenesis and that the response is similar to that obtained with FGF-2, a well-known angiogenic cytokine. It has previously been demonstrated that OC is involved in bone repair, so the angiogenic activity reported here might also play a crucial role in bone formation.     

New proangiogenic activity on vascular endothelial cells for C-terminal mechano growth factor

Angiogenesis is crucial in wound healing. The administration of the C-terminal 24-a.a. peptide of mechano growth factor (MGF24E) has been previously demonstrated to induce more blood vessels in regenerating bone around defective areas compared with the control. Accordingly, this study aims to determine whether MGF24E promotes bone defect healing through MGF24E-increased angiogenesis and whether MGF24E has positive effects on angiogenesis in vitro. The roles of MGF24E on angiogenesis and the underlying mechanisms were investigated. The cell proliferation, migration, and tubulogenesis of the human vascular endothelial EA.hy926 cells co-treated with 2% serum and MGF24E were determined to assess angiogenesis in comparison with 100 ng/ml of vascular endothelial growth factor 165 (VEGF(165))-positive control or vehicle control (phosphate-buffered saline). MGF24E treatment (10 ng/ml) significantly promoted the biological processes of angiogenesis on EA.hy926 cells compared with the vehicle control. The suppression of vascular endothelial growth factor and angiopoietin-I expressions by 2% serum starvation was reversed by the addition of 10 ng/ml of MGF24E in 2% serum medium. This result suggests that MGF24E has a protective effect on angiogenesis. Moreover, the inhibition of ERK due to PD98050 pretreatment completely abolished and mostly blocked MGF24E-induced proliferation and migration, respectively, whereas the MGF24-induced tubulogenesis and the angiogenic factor expression were only partially inhibited. These new findings suggest that MGF24E promotes angiogenesis by enhancing the expression of angiogenic cytokines which involves the MAPK/ERK-signaling pathway.     

Angiogenesis and Inflammation Signaling Are Targets of Beer Polyphenols on Vascular Cells

Emerging evidence indicates that chronic inflammation and oxidative stress cluster together with angiogenic imbalance in a wide range of pathologies. In general, natural polyphenols present health‐protective properties, which are likely attributed to their effect on oxidative stress and inflammation. Hops used in beer production are a source of polyphenols such as xanthohumol (XN), and its metabolites isoxanthohumol (IXN) and phytoestrogen 8‐prenylnaringenin (8PN). Our study aimed to evaluate XN, IXN, and 8PN effects on angiogenesis and inflammation processes. Opposite in vitro effects were observed between 8PN, stimulating endothelial and smooth muscle cell (SMC) growth, motility, invasion and capillary‐like structures formation, and XN and IXN, which inhibited them. Mouse matrigel plug and rat skin wound‐healing assays confirmed that XN and IXN treatments reduced vessel number as well as serum macrophage enzymatic activity, whereas 8PN increased blood vessels formation in both assays and enzyme activity in the wound‐healing assay. A similar profile was found for serum inflammatory interleukin‐1β quantification, in the wound‐healing assay. Our data indicate that whereas 8PN stimulates angiogenesis, XN and IXN manifested anti‐angiogenic and anti‐inflammatory effects in identical conditions. These findings suggest that the effects observed for individual compounds on vascular wall cells must be carefully taken into account, as these polyphenols are metabolized after in vivo administration. The modulation of SMC proliferation and migration is also of special relevance, given the role of these cells in many pathological conditions. Furthermore, these results may provide clues for developing useful therapeutic agents against inflammation‐ and angiogenesis‐associated pathologies. J. Cell. Biochem. 111: 1270–1279, 2010. © 2010 Wiley‐Liss, Inc.     

An assay for cancer stem cell-induced angiogenesis on chick chorioallantoic membrane

Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.     

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides

Pleiotrophin (PTN) is a heparin-binding growth/differentiation inducing cytokine that shares 50% amino acid sequence identity and striking domain homology with Midkine (MK), the only other member of the Ptn/Mk developmental gene family. The Ptn gene is expressed in sites of early vascular development in embryos and in healing wounds and its constitutive expression in many human tumors is associated with an angiogenic phenotype, suggesting that PTN has an important role in angiogenesis during development and in wound repair and advanced malignancies. To directly test whether PTN is angiogenic in vivo, we injected a plasmid to express PTN into ischemic myocardium in rats. Pleiotrophin stimulated statistically significant increases in both normal appearing new capillaries and arterioles each of which had readily detectable levels of the arteriole marker, smooth muscle cell alpha-actin. Furthermore, the newly formed blood vessels were shown to interconnect with the existent coronary vascular system. The results of these studies demonstrate directly that PTN is an effective angiogenic agent in vivo able to initiate new vessel formation that is both normal in appearance and function. The data suggest that PTN signals the more "complete" new blood vessel formation through its ability to stimulate different functions in different cell types not limited to the endothelial cell.     

The regulatory mechanism of Hsp90α secretion from endothelial cells and its role in angiogenesis during wound healing

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90α can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90α from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90α in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90α but not Hsp90β is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90α localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90α neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90α localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90α can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90α as a stimulator for wound repair.     

Plastic surgical perspectives on vascular endothelial growth factor as gene therapy for angiogenesis

The practice of plastic surgery has always remained at the frontier of medical science. Over the past few decades, this frontier has been marked by significant developments in the field of gene therapy. Gene therapy serves to replace, supplement, or manipulate a patient's genetic makeup to restore function that has been lost or to correct function that is aberrant. Recent technology may allow surgeons to augment the processes of wound healing and angiogenesis by transfecting genes encoding desirable proteins, such as vascular endothelial factor (VEGF), into ischemic tissues. VEGF is a vital growth factor in the development of blood vessels. Although its mechanisms of action are numerous, its sole function seems to be the augmentation of angiogenesis. VEGF is active in growth and development, in wound healing, and in various pathologic conditions, such as psoriasis and rheumatoid arthritis. The role of VEGF in the field of plastic surgery is just beginning to be explored; it may someday prove to be very rewarding.     

The role of fibroblast growth factor-2 in the vascularization of the chick embryo chorioallantoic membrane

The CAM is an extraembryonic membrane which serves as a gas exchange surface and its respiratory function is provided by an extensive capillary network. The development of the vascular system of the CAM is a complex, highly regulated process that depends on genetic and epigenetic factors expressed by endothelial and non-endothelial cells. In spite of the evidence that several growth factors are angiogenic in the CAM assay, poorly investigated is their role in the development of the CAM's vascular system. This article reviews our studies concerning the role of exogenous and endogenous fibroblast growth factor-2 (FGF-2) in the CAM vascularization. The findings in all these studies support the importance of FGF-2 as an autocrine paracrine stimulator of angiogenesis and its key role in the development of the vascular system in the avian embryo.     

The mouse cornea micropocket angiogenesis assay

The mouse corneal micropocket angiogenesis assay uses the avascular cornea as a canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 5 d and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor with sucralfate (a stabilizer) and Hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate)) to allow slow release). This mixture is applied to a mesh that controls unit size and then allowed to harden. A micropocket is surgically created in the mouse cornea and a pellet implanted. Five days later, the area of the cornea overgrown by the angiogenic response is measured using a slit lamp. A skilled investigator can implant and grade 40 eyes in about 2.5 h. The results of the assay are used to assess the ability of potential therapeutic molecules or genetic differences to modulate angiogenesis in vivo.     

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.