New Insights into the Role of Basement Membrane-Derived Matricryptins in the Heart.

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.

Role of pigment epithelium-derived factor in the reproductive system.

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.

Semaphorin 3F forms an anti-angiogenic barrier in outer retina.

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.

The Wnt pathway and the roles for its antagonists, DKKS, in angiogenesis.

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.

Imbalanced levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of patients with proliferative diabetic retinopathy.

A role for vascular endothelial growth factor (VEGF) has been clearly implicated in the pathogenesis of proliferative diabetic retinopathy (PDR). However, other molecules and mechanisms may be operating independently, or in conjunction with VEGF in the pathogenesis of this disease. Therefore, we made an attempt to comparatively investigate the levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of subjects with Proliferative Diabetic Retinopathy (PDR) compared to control subjects. The vitreous and plasma concentrations of VEGF, EPO (Erythropoietin) and PEDF (Pigment Epithelium Derived Factor) were measured using Enzyme Linked Immunosorbent Assay (ELISA) and the postmortem retinal tissue was subjected to Western blot analysis. The mean vitreous and plasma levels of VEGF and EPO in patients with PDR were significantly (p<0.001) higher than those in subjects without diabetes. Conversely, the vitreous and plasma levels of PEDF were significantly (p<0.001) lower in the PDR patients compared to control subjects. Multivariate logistic-regression analyses indicated that EPO was more strongly associated with PDR than VEGF. The protein expression of the VEGF and EPO in the retinal tissue was significantly higher in PDR and diabetes without complication groups compared to controls. Compared to controls, the protein expression of PEDF was significantly lower in retinal tissues from diabetes patients without complications and in patients with PDR. The fact that the vitreous and plasma levels and the retinal tissue protein expression of EPO were strongly associated with PDR implies a definite role of 'hypererythropoietinemia' in neovascularization processes.

Apoptotic potency of angiostatic compounds in the treatment of cancer.

When tumours outgrow their vascular supply, they become hypoxic because of nutrient deficiency. This increases the expression and secretion of proangiogenic factors, like vascular endothelial growth factor (VEGF), leading to the activation of endothelial cells. The activated endothelial cells migrate, proliferate and form new blood vessels, resulting in increased tumour growth. This process is called tumour angiogenesis. Inhibiting tumour angiogenesis and therefore tumour growth is a well known concept in the treatment of cancer, such as hepatocellular carcinoma (HCC). This can be done by endogenous angiogenesis inhibitors, like angiostatin and its derivates. These are known to affect endothelial cell functions including the induction of apoptosis. The impact of these angiostatic factors on the cell is manifold. This also applies for so called small molecules, which affect tyrosine kinases such as receptors or intracellular signal transduction proteins. Other approaches, like monoclonal antibodies, target a single molecule, mainly VEGF, to inhibit receptor binding and downstream signal transduction. Gene silencing, mainly via RNA interference (RNAi) intervenes on RNAlevel, leading to reduced gene expression and protein secretion. Due to intense research in this field, there is rising evidence that also tumour cells themselves are influenced by angiostatic treatment approaches and the underlying molecular mechanisms are more and more revealed. Here we give a (short) review regarding the pro-apoptotic potency of antiangiogenic compounds like angiostatic molecules, sequestering antibodies, small molecules and RNAi approaches targeting endothelial and tumour cell survival to inhibit angiogenesis and tumour growth.

Antitumor and angiostatic peptides from frog skin secretions.

The discovery of new molecules with potential antitumor activity continues to be of great importance in cancer research. In this respect, natural antimicrobial peptides isolated from various animal species including humans and amphibians have been found to be of particular interest. Here, we report the presence of two anti-proliferative peptides active against cancer cells in the skin secretions of the South American tree frog, Phyllomedusa bicolor. The crude skin exudate was fractioned by size exclusion gel followed by reverse-phase HPLC chromatography. After these two purification steps, we identified two fractions that exhibited anti-proliferative activity. Sequence analysis indicated that this activity was due to two antimicrobial α-helical cationic peptides of the dermaseptin family (dermaseptins B2 and B3). This result was confirmed using synthetic dermaseptins. When tested in vitro, synthetic B2 and B3 dermaseptins inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line by more than 90%, with an EC(50) of around 2-3 µM. No effect was observed on the growth of the NIH-3T3 non-tumor mouse cell line with Drs B2, whereas a slight inhibiting effect was observed with Drs B3 at high dose. In addition, the two fractions obtained after size exclusion chromatography also inhibited PC-3 cell colony formation in soft agar. Interestingly, inhibition of the proliferation and differentiation of activated adult bovine aortic endothelial cells was observed in cells treated with these two fractions. Dermaseptins B2 and B3 could, therefore, represent interesting new pharmacological molecules with antitumor and angiostatic properties for the development of a new class of anticancer drugs.

Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Cx37 deletion enhances vascular growth and facilitates ischemic limb recovery.

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.

Endorepellin, the angiostatic module of perlecan, interacts with both the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2): a dual receptor antagonism.

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 α2ß1 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 α2ß1 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 α2ß1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2ß1 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.

CXCL4L1 inhibits angiogenesis and induces undirected endothelial cell migration without affecting endothelial cell proliferation and monocyte recruitment.

BACKGROUND AND OBJECTIVES: The non-allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C-terminal α-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 µg 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 µg 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.

Antiangiogenic effects and transcriptional regulation of pigment epithelium-derived factor in diabetic retinopathy.

The effects of the antiangiogenic cytokine PEDF on key steps in retinal angiogenesis, specifically endothelial cell proliferation and vascular tubule formation, and the regulation of PEDF expression in retinal capillary endothelial cells were evaluated. HUVECs were co-cultured with fibroblasts to construct a model of angiogenesis using the Angiokit assay, and image analysis software was used to measure the effects of PEDF and VEGF on vascular tubule formation. Quantitative real-time PCR analysis was used to determine the expression of PEDF in microvascular endothelial cells exposed to glucose 20 mM, insulin 100 nM and VEGF 10 ng/ml. PEDF inhibited endothelial cell proliferation and significantly decreased the number of tubules (629+93 AU vs 311+31, p=0.001), number of branching points (145+19 AU vs 46+5, p=0.03) and total tubule length (4848+748 AU vs 11,172+2353, p=0.001). In bovine retinal capillary endothelial cells (BRCECs), PEDF mRNA and protein expression was suppressed by insulin (22%) in a rapamycin-sensitive manner; wortmannin had no effect. PEDF mRNA expression was also significantly reduced in the presence of high glucose (23%) and VEGF (25%). In conclusion, PEDF inhibits key steps in the angiogenic response of BRCECs, including endothelial cell proliferation and vascular tubule formation. Gene expression of PEDF is negatively regulated by glucose, insulin (via an mTOR-dependent pathway) and VEGF.

Dose-dependent effect of radiation on angiogenic and angiostatic CXC chemokine expression in human endothelial cells.

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.

Chemokines as mediators of neovascularization.

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.

Gene expression changes associated with the anti-angiogenic activity of kallikrein-related peptidase 3 (KLK3) on human umbilical vein endothelial cells.

Kallikrein-related peptidase 3 (KLK3, also known as prostate-specific antigen, PSA) is a chymotrypsin-like kallikrein that has anti-angiogenic properties. We have previously shown in a human umbilical vein endothelial cell (HUVEC) model that the anti-angiogenic effect of KLK3 is related to its enzyme activity. However, the mechanism of this effect remains to be clarified. To this end, we used a DNA microarray to study KLK3-induced changes in gene expression associated with reduction of HUVEC tube formation. Among the 41,000 genes studied, 311 were differentially expressed between control and KLK3-treated cells. These changes were enriched in several pathways, including those associated with proteasome, ubiquitin-mediated proteolysis, focal adhesion and regulation of the actin cytoskeleton. Furthermore, the changes were opposite to those previously described to occur during tubulogenesis. In conclusion, our results show that KLK3 induces gene expression changes in HUVECs. Although these changes might be relevant for the mechanism by which KLK3 exerts its anti-angiogenic activity, it cannot be judged from the present results whether they reflect the primary mechanism mediating the effect of KLK3 or are secondary to morphogenic differentiation.

Understanding the immunoangiostatic CXC chemokine network.

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.

Activation of p38(MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B.

Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.

Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead.

Sprouting angiogenesis is an invasive process that involves proteolytic activities required for the degradation of the endothelial basement membrane, cell migration with removal of obstructing matrix proteins, and generation of space in the matrix to allow endothelial cells to form a proper lumen. In the last decade it has become clear that besides these matrix-degrading properties, proteases exert additional, more subtle functions that play a key role in angiogenesis. These functions are discussed with specific emphasis on membrane type-1 matrix metalloproteinase (MT1-MMP), other MMPs, and the related ADAMs (a disintegrin and metalloproteinase domain). Proteases modulate the balance between pro- and anti-angiogenic factors by activation and modification of growth factors and chemokines, ectodomain shedding with accompanied receptor activation, shedding of cytokines from membrane-bound precursors, and generation of (matrix) protein fragments that inhibit or activate angiogenesis. Furthermore, they participate in the recruitment of leukocytes and progenitor cells, which contribute to the onset and progression of angiogenesis. Proteases facilitate the mobilization of progenitor cells in the bone marrow as well as the entry of these cells and leukocytes into the angiogenic area. The interaction between pericytes and the newly formed endothelial tubes is accompanied by silencing of MMP activities. Better understanding of the various activities of proteases may be helpful in developing more specific inhibitors that could result in tailor-made modification of proteolytic activities in disease.

Evidence for annexin II-S100A10 complex and plasmin in mobilization of cytokine activity of human TrpRS.

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.

Integrin alpha2beta1 is the required receptor for endorepellin angiostatic activity.

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.