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Background Endostatin, an angiogenic inhibitor, is associated with worse pulmonary arterial hypertension (PAH) outcomes in adults and poor lung growth in children. This study sought to assess whether endostatin is associated with disease severity and outcomes in pediatric PAH. Methods and Results Serum endostatin was measured in cross‐sectional (N=160) and longitudinal cohorts (N=64) of pediatric subjects with PAH, healthy pediatric controls and pediatric controls with congenital heart disease (CHD) (N=54, N=15), and adults with CHD associated PAH (APAH‐CHD, N=185). Outcomes, assessed by regression and Kaplan‐Meier analysis, included hemodynamics, change in endostatin over time, and transplant‐free survival. Endostatin secretion was evaluated in pulmonary artery endothelial and smooth muscle cells. Endostatin was higher in those with PAH compared with healthy controls and controls with CHD and was highest in those with APAH‐CHD. In APAH‐CHD, endostatin was associated with a shorter 6‐minute walk distance and increased mean right atrial pressure. Over time, endostatin was associated with higher pulmonary artery pressure and pulmonary vascular resistance index, right ventricular dilation, and dysfunction. Endostatin decreased with improved hemodynamics over time. Endostatin was associated with worse transplant‐free survival. Addition of endostatin to an NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide) based survival analysis improved risk stratification, reclassifying subjects with adverse outcomes. Endostatin was secreted primarily by pulmonary artery endothelial cells. Conclusions Endostatin is associated with disease severity, disease improvement, and worse survival in APAH‐CHD. Endostatin with NT‐proBNP improves risk stratification, better predicting adverse outcomes. The association of elevated endostatin with shunt lesions suggests that endostatin could be driven by both pulmonary artery flow and pressure. Endostatin could be studied as a noninvasive prognostic marker, particularly in APAH‐CHD.

In the retinal pigment epithelium (RPE) several factors within the macular compared to peripheral regions cause differences in physiological aging. The molecular mechanisms during aging in the context of topography are not well known. The proteome of RPE of different aged macular-bearing primates Callithrix jacchus was thus analysed with ion mobility mass spectrometry. Macular and periphery of neonate RPE were well differentiated from aged tissues as demonstrated by principal component analysis. This finding was mainly due to proteins involved in major developmental processes and the visual cycle. The distinction of adult from senile tissue and macular from periphery was more subtle. The hypotheses of inflammation increasing with age was supported. High expression levels of proteins related to oxidative stress (e.g., cathepsin B) and chaperones (e.g., HSP90) were detected in aged RPE as confirmed by Western blot and immunohistochemical analysis. Decreased levels of proteins participating in angiostatic properties (e.g., thrombospondin 1) and the integrity of tissue basement membranes with age (e.g., nidogen 1) were in agreement with neovascularization. This study presents targets for further investigations of the mechanisms of the aging process with the aim to elucidate predictive factors for the conversion of physiological aging into pathological conditions. Significance The current study characterized the different protein profiles of the retinal pigment epithelium (RPE) of the macula-bearing, non-human primate Callithrix jacchus during life-time. In addition, the subproteomes of macular and peripheral RPE were investigated. Differently expressed proteins described developmental processes in neonate tissue and destructive mechanisms in aged samples. Insights into the physiological aging process of the RPE and its conversion into pathophysiological conditions were gained. They assist in designing therapeutical approaches to counteract age-related diseases of the retina.

The extracellular matrix (ECM), which contributes to structural homeostasis as well as to the regulation of cellular function, is enzymatically cleaved by proteases, such as matrix metalloproteinases and cathepsins, in the normal and diseased heart. During the past two decades, matricryptins have been defined as fragments of ECM with a biologically active cryptic site, namely the 'matricryptic site,' and their biological activities have been initially identified and clarified, including anti-angiogenic and anti-tumor effects. Thus, matricryptins are expected to be novel anti-tumor drugs, and thus widely investigated. Although there are a smaller number of studies on the expression and function of matricryptins in fields other than cancer research, some matricryptins have been recently clarified to have biological functions beyond an anti-angiogenic effect in heart. This review particularly focuses on the expression and function of basement membrane-derived matricryptins, including arresten, canstatin, tumstatin, endostatin and endorepellin, during cardiac diseases leading to heart failure such as cardiac hypertrophy and myocardial infarction.

The physiological function of the female reproductive organs is hormonally controlled. In each cycle, the reproductive organs undergo tissue modifications that are accompanied by formation and destruction of blood vessels. Proper angiogenesis requires an accurate balance between stimulatory and inhibitory signals, provided by pro- and anti-angiogenic factors. As with many other tissues, vascular endothelial growth factor (VEGF) appears to be one of the major pro-angiogenic factors in the female reproductive organs. Pigment epithelium-derived factor (PEDF) is a non-inhibitory member of the serine protease inhibitors (serpin) superfamily, possessing potent physiologic anti-angiogenic activity that negates VEGF activity. The role of PEDF in decreasing abnormal neovascularization by exerting its anti-angiogenic effect that inhibits pro-angiogenic factors, including VEGF, has been investigated mainly in the eye and in cancer. This review summarizes the function of PEDF in the reproductive system, showing its hormonal regulation and its anti-angiogenic activity. Furthermore, some pathologies of the female reproductive organs, including endometriosis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, and others, are associated with a faulty angiogenic process. This review illuminates the role of PEDF in their pathogenesis and treatment. Collectively, we can conclude that although PEDF seems to play an essential role in the physiology and pathophysiology of the reproductive system, its full role and mechanism of action still need to be elucidated.

The Wnt signaling pathway is involved in a wide range of developmental and physiological processes, such as cell fate specification, tissue morphogenesis, and homeostasis. Thus, its dysregulation has been found in multiple diseases, including some cardiovascular disorders. The loss or gain of function of Wnt pathway components results in abnormal vascular development and angiogenesis. Further study has revealed that Wnt signaling in endothelial cells appears to contribute to vascular morphogenesis and endothelial cell specification. Owing to the significance of Wnt signaling in angiogenesis, Wnt antagonists have been considered potential treatments for neovascular disorders. In line with this, members of the Dkk protein family (Dkks), well-known Wnt antagonists, have been recently found to regulate angiogenesis. This review summarizes our present knowledge of the roles of Wnt signaling and Wnt antagonists, particularly Dkks, in angiogenic regulation and explores the therapeutic potential of Wnt antagonists.

background: Angiogenesis, i.e. the development of new blood vessels from pre-existing ones, represents an integral part in the patho- genesis of endometriosis. During the last decade, an increasing number of studies have therefore focused on the anti-angiogenic treatment of the disease. The present review provides a systematic overview of these studies and critically discusses the future role of anti-angiogenic therapy in the multimodal management of endometriosis. methods: Literature searches were performed in PubMed, MEDLINE and ISI Web of Knowledge for original articles published before the end of March 2012, written in the English language and focusing on anti-angiogenic approaches for the therapy of endometriosis. The searches included both animal and human studies. results: Numerous compounds of different substance groups have been shown to exert anti-angiogenic effects on endometriotic lesions under experimental in vitro and in vivo conditions. These include growth factor inhibitors, endogenous angiogenesis inhibitors, fumagillin analogues, statins, cyclo-oxygenase-2 inhibitors, phytochemical compounds, immunomodulators, dopamine agonists, peroxisome prolifer- ator-activated receptor agonists, progestins, danazol and gonadotropin-releasing hormone (GnRH) agonists. However, clinical evidence for their efficacy in anti-angiogenic endometriosis therapy is still lacking. conclusions: Anti-angiogenic compounds hold great promise for the future treatment of endometriosis because they may inhibit the establishment of new endometriotic lesions in early stages of the disease or after surgical treatment. Further experimental studies, controlled clinical trials in particular, are required now to clarify which compounds fulfil these expectations without inducing severe side effects in patients with endometriosis.

Matrix metalloproteinase-2 (MMP-2) degrades type IV collagen and enables endothelial cell (EC) migration during angiogenesis and wound healing. Peroxisomal biogenesis factor 2 (PEX2), a by-product of activated MMP-2 autocatalysis, competitively inhibits newly activated MMP-2 from EC surface binding and migration. We hypothesize that PEX2 is elevated during limb ischemia and contributes to poor wound healing, with decreased capillary density.Western blot was used to identify PEX2 in the hind limbs of FVB/NJ mice with surgically induced ischemia. The PEX2 effect on healing was evaluated by calculating the area of exposed muscle after wounding the dorsum of mice and administering daily injections with human recombinant PEX2 (hrPEX2). Wounds were also injected with lentivirus-expressing PEX2 (PEX2-LV), harvested on postoperative day 7 and processed for staining. Epithelial gap was assessed with light microscopy. Capillary density was evaluated after wounding Tie2-green fluorescent protein (GFP)(+) transgenic FVB mice (ECs labeled green) and viral transduction with PEX2-LV. Wounds were harvested on postoperative day (POD) 7, frozen in liquid nitrogen, sectioned, and stained with Hoechst. Vessel density was assessed via fluorescence microscopy as the average number of capillaries/10 high-powered fields. Paired t test was used to assess differences between the groups.PEX2 was elevated 5.5 ± 2.0-fold (P = .005) on POD 2 and 2.9 ± 0.69-fold (P = .004) on POD 4 in gastrocnemius muscles of ischemic hind limbs. The wound surface area, or lack of granulation tissue and exposed muscle, decreased daily in all mice but was greater in the hrPEX2-treated mice by 12% to 16% (P.004). Wounds in the control group were completely covered with granulation tissue by POD 3. Wounds injected with hrPEX2 were not completely covered by POD 7 but continued to have exposed muscle. Microscopic examination of wounds after PEX2-LV viral transduction demonstrated an average epithelial gap of 1.6 ± 0.3 vs 0.64 ± 0.3 μm in control wounds (P.04). Wounds from Tie2-GFP mice had an average number of 3.8 ± 1.1 capillaries vs 6.9 ± 1.2 in control wounds (P.007).Our study links elevated PEX2 to ischemia and poor wound healing. We demonstrate comparative PEX2 elevation in ischemic murine hind limbs. Less granulation tissue is produced and healing is retarded in wounds subjected to hrPEX2 or viral transduction with PEX2-LV. Microscopic examination shows the wounds exhibit fewer capillaries, supporting the hypothesis that PEX2 decreases angiogenesis.

The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37−/−) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40−/− animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37−/− animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37−/− mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37−/− animals compared with WT animals. Cx37−/− mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37−/− animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.

The 16-kDa angiostatic N-terminal fragment of human prolactin (16K hPRL) has been reported to be a new potent anticancer compound. This protein has already proven its efficiency in several mouse tumor models in which it prevented tumor-induced angiogenesis and delayed tumor growth. In addition to angiogenesis, tumors also stimulate the formation of lymphatic vessels, which contribute to tumor cell dissemination and metastasis. However, the role of 16K hPRL in tumor-induced lymphangiogenesis has never been investigated. We establish in vitro that 16K hPRL induces apoptosis and inhibits proliferation, migration, and tube formation of human dermal lymphatic microvascular endothelial cells. In addition, in a B16F10 melanoma mouse model, we found a decreased number of lymphatic vessels in the primary tumor and in the sentinel lymph nodes after 16K hPRL treatment. This decrease is accompanied by a significant diminished expression of lymphangiogenic markers in primary tumors and sentinel lymph nodes as determined by quantitative RT-PCR. These results suggest, for the first time, that 16K hPRL is a lymphangiostatic as well as an angiostatic agent with antitumor properties.

Background and Objectives: The non-allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C-terminal alpha-helix and has been characterized as a potent anti-angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a 'classical' chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti-proliferative activities, and proinflammatory functions, which can be conferred by heteromer-formation with CCL5/RANTES enhancing monocyte recruitment. Methods and Results: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC(50) 6.9 mu g mL-1) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin-neutralizing activity than CXCL4 (IC(50) 2.45 vs 0.98 mu g mL-1). Conclusions: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.

This prospective study was designed to determine whether variation in angiogenic (placental growth factor [PlGF]) and/or anti-angiogenic (soluble fms-like tyrosine kinase [sFlt-1]) factors contribute to the protective effect of highland ancestry (Andean) from altitude-associated reductions in fetal growth.Plasma sFlt-1 and PlGF levels, uterine artery (UA) blood flow, and fetal biometry were determined in low-altitude (400 m; Andean n = 27, European n = 28) and high-altitude (3600 m; Andean n = 51, European n = 44) residents during pregnancy (20 and 36 weeks) and 4 months postpartum.High-altitude decreased sFlt-1 levels in both groups, Andeans had lower sFlt-1, comparable PlGF, lower sFlt-1/PlGF ratios, and higher UA blood flow throughout pregnancy relative to Europeans. Altitude decreased birth weight in Europeans but not Andeans. In high-altitude Europeans sFlt-1/PlGF and sFlt-1 levels were negatively associated with UA diameter and birth weight, respectively.Lower sFlt-1 and sFlt-1/PLGF ratio may contribute to or result from variations in maternal vascular adaptation to pregnancy between Andean and Europeans at high altitude. Subsequently, these effects could potentially influence ancestry-associated differences in birth weight.

Chemokines are a superfamily of homologous heparin-binding proteins, first described for their role in recruiting leukocytes to sites of inflammation. Chemokines have since been recognized as key factors mediating both physiological and pathological neovascularization in such diverse clinical settings as malignancy, wound repair, chronic fibroproliferative disorders, myocardial ischemia, and atherosclerosis. Members of the CXC chemokine family, structurally defined as containing the ELR amino acid motif, are potent inducers of angiogenesis, whereas another subset of the CXC chemokines inhibits angiogenesis. In addition, CCL2, a CC chemokine ligand, has been implicated in arteriogenesis. In this article, we review the current literature on the role of chemokines as mediators of neovascularization.

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.

In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1β and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1β was enhanced synergistically by TNF-α but inhibited by IFN-γ, which synergized with IL-1β to produce the angiostatic CXCL10/IFN-γ-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.

Angiogenesis plays an important role in the growth and progression of cancer. The regulation of tumor angiogenesis depends on a net balance of angiogenic factors and antiangiogenic factors, which are secreted by both tumor cells and host-infiltrating cells. Numerous studies have indicated that assessment of angiogenic activity by either microvessel density or expression of angiogenic factors in cancer can provide prognostic information independent of conventional clinicopathological factors such as tumor staging. Some studies also suggested that assessment of tumor angiogenesis may predict cancer response to chemotherapy or radiotherapy. However, the most important clinical implication of tumor angiogenesis is the development of a novel strategy of anticancer therapy targeting tumor vessels instead of cancer cells. Antiangiogenic therapy aims to inhibit the growth of tumor, and current evidence suggests that it works best in combination with conventional cytotoxic chemotherapy. Recently, a monoclonal antibody against vascular endothelial growth factor, which is one of the most potent angiogenic factors, has been approved for clinical use in colorectal cancer patients after a clinical trial confirmed that combining the antibody with standard chemotherapy regimen could prolong patient survival. The clinical implications of angiogenesis in cancer are reviewed in this article.

In systemic sclerosis (SSc), clinical evidence has shown that Bosentan may foster the regeneration of the peripheral microcirculatory network. The aim of this study was to verify in vitro the influence of Bosentan on the angiogenic performance of dermal microvascular endothelial cells (MVECs) and its possible capacity to counteract the antiangiogenic effects of SSc sera.Healthy dermal MVECs were challenged with Bosentan at different concentrations (0.1 μM, 1 μM, 10 μM) or with sera from patients with diffuse cutaneous SSc (n=8) and healthy subjects (n=8), alone or in combination with Bosentan (10 μM). Cell viability and chemoinvasion were determined by WST-1 and Boyden chamber assays, respectively. Angiogenesis was evaluated by capillary morphogenesis on Matrigel.Challenge of dermal MVECs with SSc sera induced a significant reduction in angiogenesis (p0.005 vs. basal condition; p0.001 vs. healthy sera). The addition of Bosentan could significantly restore angiogenesis in the presence of SSc sera (p0.01 vs. SSc sera alone). Healthy sera promoted cell viability which was, instead, significantly reduced with SSc sera (p0.005 vs. healthy sera). The addition of Bosentan to MVECs challenged with SSc sera significantly increased cell viability (p0.005 vs. SSc sera alone), reaching levels similar to MVECs treated with healthy sera. Co-incubation of MVECs with Bosentan and SSc sera significantly increased chemoinvasion (p0.005 vs. SSc sera alone) which was inhibited by SSc sera (0.001 vs. healthy sera).Bosentan effectively counteracts the antiangiogenic effects of SSc sera on dermal MVECs and fosters the restoration of a proangiogenic environment.

Endorepellin, the C-terminal domain of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity. The terminal laminin-like globular (LG3) domain of endorepellin appears to possess most of the biological activity on endothelial cells. LG3 protein has been detected in the urine of patients with end-stage renal disease and in the amniotic fluid of pregnant women with premature rupture of fetal membranes. These findings suggest that proteolytic processing of endorepellin and the generation of LG3 might have biological significance. In this study, we have identified specific enzymes of the bone morphogenetic protein-1 (BMP-1)/Tolloid family of metalloproteases that cleave LG3 from recombinant endorepellin at the physiologically relevant site and that cleave LG3 from endogenous perlecan in cultured mouse and human cells. The BMP-1/Tolloid family of metalloproteases is thereby implicated in the processing of a major basement membrane proteoglycan and in the liberation of an anti-angiogenic factor. Using molecular modeling, site-directed mutagenesis and angiogenic assays, we further demonstrate that LG3 activity requires specific amino acids involved in Ca(2+) coordination.

// Katrien Van Raemdonck 1 , Nele Berghmans 1 , Vincent Vanheule 1 , Antonella Bugatti 2 , Paul Proost 1 , Ghislain Opdenakker 3 , Marco Presta 2 , Jo Van Damme 1 , Sofie Struyf 1 1 Laboratory of Molecular Immunology, KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Leuven, Belgium 2 Laboratory of Experimental Oncology and Immunology, University of Brescia, Department of Molecular and Translational Medicine Brescia, Italy 3 Laboratory of Immunobiology, KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Leuven, Belgium Correspondence to: Sofie Struyf, e-mail: Sofie.Struyf@rega.kuleuven.be Keywords: chemokines, CXCL4, CXCL4L1, EGF, CCL5, angiogenesis Received: June 12, 2014 Accepted: September 29, 2014 Published: October 21, 2014 ABSTRACT CXCL4 and CXCL4L1, platelet-derived CXC chemokines, and their carboxy-terminal peptides CXCL4 47–70 and CXCL4L1 47–70 previously displayed angiostatic and anti-tumoral activity in a melanoma model. Here, we found CXCL4 47–70 and CXCL4L1 47–70 to inhibit lymphatic endothelial cell proliferation in vitro . Furthermore, the angiostatic potential of CXCL4 47–70 and CXCL4L1 47–70 was tested against different angiogenic stimuli (FGF1, FGF2, FGF8, EGF and VEGF). Besides reducing FGF2-induced vascular endothelial cell growth, CXCL4 47–70 and CXCL4L1 47–70 efficiently counteracted EGF. Consequently, we considered their anti-tumoral potential in EGF-dependent MDA-MB-231 breast tumors. In tumor-bearing mice, CXCL4 47–70 reduced tumor growth better than CXCL4L1 47–70 . In CXCL4 47–70 -treated tumors significantly more intratumoral monocytes/macrophages and dendritic cells were present and higher expression levels of CCL5 and IFN- γ were detected by qPCR on tumor lysates. Because neither peptide was able to specifically bind CXCR3A or CXCR3B, differential glycosaminoglycan binding and direct interaction with cytokines (EGF and CCL5) might explain any differences in anti-tumoral effects. Notably, CCL5-induced monocyte chemotaxis in vitro was increased by addition of CXCL4 47–70 or CXCL4L1 47–70 . Finally, CXCL4 47–70 and CXCL4L1 47–70 inhibited proliferation of MDA-MB-231 cells. Our results suggest a tumor type-dependent responsiveness to either CXCL4 47–70 or CXCL4L1 47–70 treatment, defined by anti-proliferative, angiostatic and inflammatory actions, and substantiate their therapeutic potential.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrosing interstitial pneumonia of unknown aetiology that usually leads to respiratory failure and death within 5 years of diagnosis. Alveolar epithelial cell injury, disruption of alveolar capillary membrane integrity and abnormal vascular repair and remodelling have all been proposed as possible pathogenic mechanisms. This review summarizes our current knowledge of the abnormalities in vascular remodelling observed in IPF and highlights several of the cytokines thought to play a pathogenic role, which may ultimately prove to be future therapeutic targets.

Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2β1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2β1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2β1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2β1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.

Purpose: Tumor-released proangiogenic factors suppress endothelial adhesion molecule (EAM) expression and prevent leukocyte extravasation into the tumor. This is one reason why immunotherapy has met with limited success in the clinic. We hypothesized that overcoming EAM suppression with angiogenesis inhibitors would increase leukocyte extravasation and subsequently enhance the effectiveness of cellular immunotherapy. Experimental Design: Intravital microscopy, multiple color flow cytometry, immunohistochemistry, and various tumor mouse (normal and T-cell deficient) models were used to investigate the temporal dynamics of cellular and molecular events that occur in the tumor microenvironment during tumor progression and angiostatic intervention. Results: We report that while EAM levels and T-cell infiltration are highly attenuated early on in tumor growth, angiostatic therapy modulates these effects. In tumor models with normal and T-cell–deficient mice, we show the active involvement of the adaptive immune system in cancer and differentiate antiangiogenic effects from antiangiogenic mediated enhancement of immunoextravasation. Our results indicate that a compromised immune response in tumors can be obviated by the use of antiangiogenic agents. Finally, with adoptive transfer studies in mice, we show that a phased combination of angiostatic therapy and T-cell transfer significantly (P < 0.0013) improves tumor growth inhibition. Conclusions: This research contributes to understand the cellular mechanism of action of angiostatic agents and the immune response within the tumor microenvironment, in particular as a consequence of the temporal dynamics of EAM levels. Moreover, our results suggest that adjuvant therapy with angiogenesis inhibitors holds promise for cellular immunotherapy in the clinic. Clin Cancer Res; 17(10); 3134–45. ©2011 AACR.

Chemokines influence tumor growth directly or indirectly via both angiogenesis and tumor-leukocyte interactions. Platelet factor-4 (CXCL4/PF-4), which is released from α-granules of activated platelets, is the first described angiostatic chemokine. Recently, it was found that the variant of CXCL4/PF-4 (CXCL4L1/PF-4var) could exert a more pronounced angiostatic and antitumoral effect than CXCL4/PF-4. However, the molecular mechanisms of the angiostatic activities of the PF-4 forms remain partially elusive. Here, we studied the biological properties of the chemically synthesized COOH-terminal peptides of CXCL4/PF-4 (CXCL4/PF-447-70) and CXCL4L1/PF-4var (CXCL4L1/PF-4var47-70). Both PF-4 peptides lacked monocyte and lymphocyte chemotactic activity but equally well inhibited (25 nmol/L) endothelial cell motility and proliferation in the presence of a single stimulus (i.e., exogenous recombinant fibroblast growth factor-2). In contrast, when assayed in more complex angiogenesis test systems characterized by the presence of multiple mediators, including in vitro wound-healing (2.5 nmol/L versus 12.5 nmol/L), Matrigel (60 nmol/L versus 300 nmol/L), and chorioallantoic membrane assays, CXCL4L1/PF-4var47-70 was found to be significantly (5-fold) more angiostatic than CXCL4/PF-447-70. In addition, low (7 μg total) doses of intratumoral CXCL4L1/PF-4var47-70 inhibited B16 melanoma growth in mice more extensively than CXCL4/PF-447-70. This antitumoral activity was predominantly mediated through inhibition of angiogenesis (without affecting blood vessel stability) and induction of apoptosis, as evidenced by immunohistochemical and fluorescent staining of B16 tumor tissue. In conclusion, CXCL4L1/PF-4var47-70 is a potent antitumoral and antiangiogenic peptide. These results may represent the basis for the design of CXCL4L1/PF-4var COOH-terminal–derived peptidomimetic anticancer drugs. Mol Cancer Res; 8(3); 322–34

Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein–coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)–dependent G1-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G1-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a Gi-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through Gi, anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of Gi and unaffected by anastellin. These results suggest that lysophospholipid mediators of Gi activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of Gi and that anastellin selectively inhibits the Ras/ERK arm of the pathway. (Mol Cancer Res 2009;7(2):255–65)

Blood vessel growth is regulated by angiogenic and angiostatic CXC chemokines, and radiation is a vasculogenic stimulus. We investigated the effect of radiation on endothelial cell chemokine signaling, receptor expression, and migration and apoptosis. Human umbilical vein endothelial cells were exposed to a single fraction of 0, 5, or 20 Gy of ionizing radiation (IR). All vasculogenic chemokines (CXCL1–3/5–8) increased 3–13-fold after 5 or 20 Gy IR. 20 Gy induced a marked increase (1.6–4-fold) in angiostatic CXC chemokines. CXCR4 expression increased 3.5 and 7-fold at 48 h after 5 and 20 Gy, respectively. Bone marrow progenitor cell chemotaxis was augmented by conditioned media from cells treated with 5 Gy IR. Whereas 5 Gy markedly decreased intrinsic cell apoptosis (0 Gy = 16% ± 3.6 vs. 5 Gy = 4.5% ± 0.3), 20 Gy increased it (21.4% ± 1.2); a reflection of pro-survival angiogenic chemokine expression. Radiation induces a dose-dependent increase in pro-angiogenic CXC chemokines and CXCR4. In contrast, angiostatic chemokines and apoptosis were induced at higher (20 Gy) radiation doses. Cell migration improved significantly following 5 Gy, but not 20 Gy IR. Collectively, these data suggest that lower doses of IR induce an angiogenic cascade while higher doses produce an angiostatic profile.

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells. HDAC6 inhibitor tubacin, and knockdown of HDAC6, also lead to ADAMTS1 upregulation. By reporter, DAPA, and ChIP assays, the proximal GC boxes were demonstrated to be essential for ADAMTS1 induction. Decreased binding of SP1 and HDAC6 to the ADAMTS1 promoter after TSA treatment was also seen. These data suggest the involvement of HDAC6 and SP1 in the HDACi-induced expression of angiostatic ADAMTS1.

Chemokines, originally discovered as mediators of directional migration of immune cells to sites of inflammation and injury, have a function beyond their role in leukocyte chemotaxis. Indeed, they participate in organ development, angiogenesis, tumourigenesis and, more importantly, in the immune response. The chemokine family characterized by four highly conserved cysteine amino acid residues, with two cysteine residues (C) and a non-cysteine amino acid (X) between them (CXC), is known for its ability to promote trafficking of various leukocytes and to regulate angiogenesis and vascular remodelling. Intriguingly, the presence or absence of a structural-functional domain constituted by glutamic acid-leucine-arginine motif that precedes the first cysteine amino acid residue accounts for their unique property to induce or inhibit angiogenesis (angiogenic or angiostatic activity). The ability of CXC chemokine receptor 3 to promote Th1-dependent immunity and, at the same time, inhibit angiogenesis (immunoangiostasis) is of critical importance for inducing tumour regression. Agents that are able to inhibit angiogenic activities or promote angiostatic activities of CXC chemokines are future targets for research on cancer treatment. Here, we review insights on CXC chemokines in the context of immunoangiostasis and vascular damage.

The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent angiostatic factor that inhibits tumor growth in mouse models. Using microarray experiments, we have dissected how the endothelial-cell genome responds to 16K hPRL treatment. We found 216 genes that show regulation by 16K hPRL, of which a large proportion turned out to be associated with the process of immunity. 16K hPRL induces expression of various chemokines and endothelial adhesion molecules. These expressions, under the control of nuclear factor-kappaB, result in an enhanced leukocyte-endothelial cell interaction. Furthermore, analysis of B16-F10 tumor tissues reveals a higher expression of adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, or E-selectin) in endothelial cells and a significantly higher number of infiltrated leukocytes within the tumor treated with 16K hPRL compared with the untreated ones. In conclusion, this study describes a new antitumor mechanism of 16K hPRL. Because cellular immunity against tumor cells is a crucial step in therapy, the discovery that treatment with 16K hPRL overcomes tumor-induced anergy may become important for therapeutic perspectives.

Endorepellin, a C-terminal fragment of the vascular basement membrane proteoglycan perlecan, inhibits angiogenesis via the α2β1-integrin receptor. Because this integrin is also implicated in platelet-collagen responses and because endorepellin or its fragments are generated in response to injury and inflammation, we hypothesized that endorepellin could also affect platelet biology. We discovered that endorepellin supported α2β1-dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an α2β1-integrin function-blocking antibody. Collectively, these results suggest that endorepellin/α2β1-integrin interaction and effects are specific and dependent on cell type, differ from those emanated by exposure to collagen, and may be due to cellular differences in α2β1-integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic basement membrane fragments in platelet biology.

PURPOSE. A carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS) has demonstrated potent angiostatic activity during retinal developmental neovascularization in vivo. The effects of T2-TrpRS on pathologic neovascularization were tested and compared with a potent VEGF antagonist using the mouse model of oxygen-induced retinopathy (OIR). METHODS. C57BL/6J mice were transiently exposed to hyperoxic conditions (75% O2) between postnatal day 7 (P7) and P12 and then returned to room air. Retinas were isolated, blood vessels stained with isolectin Griffonia simplicifolia, images of retinal whole-mounts acquired, and the area of vascular obliteration and extent of preretinal neovascularization quantified. This method was compared to the commonly used method of OIR quantification in which the number of pre‐ inner limiting membrane (ILM) nuclei is counted in serial sections of whole eyes. To assess the angiostatic activity of T2-TrpRS, mice were injected intravitreally at P12 with either T2-TrpRS, a VEGF aptamer, or vehicle (PBS) alone, and the effects on area of obliteration and on preretinal neovascular tuft formation were assessed. RESULTS. Using a modified method of quantification in the mouse OIR model based on images of isolectin-stained retinal wholemounts, we were able to assess reliably and consistently both vascular obliteration and preretinal neovascular tuft formation in the same specimen. T2-TrpRS demonstrated potent angiostatic activity, reducing the appearance of pathologic neovascular tufts by up to 90%. Surprisingly, T2-TrpRS also enhanced physiological revascularization of the obliterated retinal vasculature, reducing these areas by up to 60% compared with PBS-injected eyes. In contrast, the VEGF antagonist, while similarly reducing preretinal neovascular tuft formation, did not enhance revascularization of the obliterated areas. CONCLUSIONS. Use of a rapid, quantifiable method to assess the effect of T2-TrpRS on retinal angiogenesis in the OIR model demonstrates the importance of a quantification system that permits simultaneous analysis of a drug’s effect on vascular obliteration as well as on preretinal neovascularization. The results obtained using this method suggest enhanced clinical value for compounds such as T2-TrpRS that not only inhibit pathologic neovascularization, but also facilitate physiological revascularization of ischemic tissue. (Invest Ophthalmol Vis Sci. 2006;47:2125‐2134) DOI:10.1167/iovs.05-1096

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

The immune system can both promote and suppress cancer. Chronic inflammation and proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are considered to be tumour promoting. In contrast, the exact nature of protective antitumour immunity remains obscure. Here, we quantify locally secreted cytokines during primary immune responses against myeloma and B-cell lymphoma in mice. Strikingly, successful cancer immunosurveillance mediated by tumour-specific CD4+ T cells is consistently associated with elevated local levels of both proinflammatory (IL-1α, IL-1β and IL-6) and T helper 1 (Th1)-associated cytokines (interferon-γ (IFN-γ), IL-2 and IL-12). Cancer eradication is achieved by a collaboration between tumour-specific Th1 cells and tumour-infiltrating, antigen-presenting macrophages. Th1 cells induce secretion of IL-1β and IL-6 by macrophages. Th1-derived IFN-γ is shown to render macrophages directly cytotoxic to cancer cells, and to induce macrophages to secrete the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10. Thus, inflammation, when driven by tumour-specific Th1 cells, may prevent rather than promote cancer., Inflammation can result in the formation of tumours, but the immune system is also involved in the elimination of cancer cells. Here, the authors show that inflammation driven by tumour-specific CD4+ T cells results in tumour regression and identify a list of cytokines associated with cancer prevention.

Objective. To investigate serum levels of a panel of angiogenic inducers (VEGF, FGF-2, Angiopoietin 1, -2, soluble VCAM-1) and inhibitors (angiostatin, endostatin, pentraxin-3) in patients with giant cell arteritis (GCA) and Takayasu’s arteritis (TAK), in order to gain further insights into the molecular mechanisms driving angiogenesis dysregulation in large-vessel vasculitis (LVV). Methods. Sera were obtained from 33 TAK patients and 14 GCA patients and from two groups of age-matched normal controls (NC). Disease activity was assessed using 18F-FDG PET/CT and clinical indices including NIH/ Kerr criteria and ITAS. Angiogenic and anti-angiogenic factor serum levels were evaluated using commercial ELISA kits. Pentraxin 3 (PTX3) serum levels were evaluated by non-commercial ELISA, as already described. Results. Among the angiogenic factors, only VEGF serum levels were significantly higher in TAK patients compared to NC. No difference was found between angiogenic factor levels in GCA patients compared to those detected in NC. Anti-angiogenic factor (Angiostatin, Endostatin, PTX3) serum levels were significantly higher in both GCA and TAK patients compared to NC. Significant associations were observed between VEGF and PTX3 levels and disease activity evaluated using PET scan and clinical indices. Cluster analysis based on PET scan scores in TAK patients showed significant ordered differences in VEGF and angiostatin serum levels. Indeed, we noted a progressive increase of VEGF and angiostatin from NC to the cluster including patients with the highest and more diffuse scan positivity. Conclusion. Our overall results demonstrate a circulating molecular profile characterised by a prevailing expression of anti-angiogenic soluble factors.