The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages

The purpose of this study was to define early events during the angiogenic response of the aortic wall to injury. Rat aortic rings produced neovessels in collagen culture but lost this capacity over time. These quiescent rings responded to vascular endothelial growth factor but not to a mixture of macrophage-stimulatory cytokines and chemokines that was angiogenically active on fresh rings. Analysis of cytokine receptor expression revealed selective loss in quiescent rings of the proangiogenic chemokine receptor CXCR2, which was expressed predominantly in aortic macrophages. Pharmacologic inhibition of CXCR2 impaired angiogenesis from fresh rings but had no effect on vascular endothelial growth factor-induced angiogenesis from quiescent explants. Angiogenesis was also impaired in cultures of aortic rings from CXCR2-deficient mice. Reduced CXCR2 expression in quiescent rat aortic rings correlated with marked macrophage depletion. Pharmacologic ablation of macrophages from aortic explants blocked formation of neovessels in vitro and reduced aortic ring-induced angiogenesis in vivo. The angiogenic response of macrophage-depleted rings was completely restored by adding exogenous macrophages. Moreover, angiogenesis from fresh rings was promoted by macrophage CSF (CSF-1) and inhibited with anti-CSF-1 Ab. Thus, aortic angiogenic sprouting following injury is strongly influenced by conditions that modulate resident macrophage numbers and function.     

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce.PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway.MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis.ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1.ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.     

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation

Membrane type 1-matrix metalloprotease (MT1-MMP or MMP-14) is a major activator of pro-MMP-2 and is essential for skeletal development. We show here that it is required for branching morphogenesis of the submandibular gland but not the lung. Instead, in the lung, it is essential for postnatal development of alveolar septae. Lung development in Mmp14-/- mice is arrested at the prealveolar stage with compensatory hyperinflation of immature saccules. Mmp2-/- mice lacked comparable defects in the lung and submandibular gland, suggesting that MT1-MMP acts via mechanisms independent of pro-MMP-2 activation. Since the developmental defects in the lung are first manifest around the time of initial vascularization (E16.5), we investigated the behavior of pulmonary endothelial cells from Mmp14+/+ and Mmp14-/- mice. Endothelial cells from lungs of 1-week-old Mmp14-/- mice show reduced migration and formation of three-dimensional structures on Matrigel. Since pulmonary septal development requires capillary growth, the underlying mechanism of pulmonary hypoplasia in Mmp14-/- mice may be defective angiogenesis, supporting a model in which angiogenesis is a critical rate-limiting step for acquisition of pulmonary parenchymal mass.     

Attenuation of retinal vascular development and neovascularization in PECAM-1-deficient mice

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1−/−) mice. A significant decrease in retinal vascular density was observed in PECAM-1−/− mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1−/− mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1−/− mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1−/− mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1−/− mice. In addition, retinal neovascularization was attenuated in PECAM-1−/− mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1−/− mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.     

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.     

Angiogenesis

Extracellular matrix (ECM) is essential for all stages of angiogenesis. In the adult, angiogenesis begins with endothelial cell (EC) activation, degradation of vascular basement membrane, and vascular sprouting within interstitial matrix. During this sprouting phase, ECM binding to integrins provides critical signaling support for EC proliferation, survival, and migration. ECM also signals the EC cytoskeleton to initiate blood vessel morphogenesis. Dynamic remodeling of ECM, particularly by membrane-type matrix metalloproteases (MT-MMPs), coordinates formation of vascular tubes with lumens and provides guidance tunnels for pericytes that assist ECs in the assembly of vascular basement membrane. ECM also provides a binding scaffold for a variety of cytokines that exert essential signaling functions during angiogenesis. In the embryo, ECM is equally critical for angiogenesis and vessel stabilization, although there are likely important distinctions from the adult because of differences in composition and abundance of specific ECM components.     

Metabolite control of angiogenesis: angiogenic effect of citrate

Endothelial cells respond to hypoxic changes with resultant accumulation of several metabolites and switch over to angiogenic phenotype. Although certain intermediates of glycolytic and oxidative metabolic pathways have been known to affect angiogenesis, the effect of citrate, which accumulates in certain tumors, on angiogenesis is not known. Therefore, the effect of citrate on angiogenesis was studied using different model systems. Increased vascularization in chorioallantoic membrane assay, increased endothelial sprouting in rat aortic rings, and increased expression of CD31, E-selectin in endothelial cells suggested a possible proangiogenic effect of citrate. Upregulation of angiogenic factors such as vascular endothelial growth factor and fibroblast growth factor suggested that the effect of citrate involves modulation of expression of angiogenic growth factors. LY 294002, an inhibitor of PI3K–Akt pathway, and wortmannin, an inhibitor of Akt pathway, reversed the effect of citrate in human umbilical vein endothelial cells. Citrate induced significant upregulation and activation of Akt in endothelial cells. Rapamycin, an inhibitor of mTOR, also reversed the effect of citrate in human umbilical vein endothelial cells and sprouting of aortic rings suggesting that the angiogenic effect of citrate involves activation of PI3K–Akt–mTOR pathway.     

Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis

Plasminogen activator (PA) inhibitor-1 (PAI-1) has been recognized as a surrogate marker of endothelial dysfunction in diseases associated with impaired angiogenesis, including atherosclerosis, diabetic vasculopathy, and nephropathy. To establish the necessary and sufficient components of the PA system [PAI-1, urokinase-type PA (uPA), or tissue-type PA (tPA), and plasminogen (Plg)] for angiogenesis, we examined angiogenic competence of vascular explant cultures obtained from mice deficient in PAI-1, tPA, uPA, and Plg. To gain insight into the requirement for different matrix-degrading systems during endothelial cell migration across plasmin-degradable basement membranes compared with profibrotic areas containing plasmin-nondegradable collagen, we contrasted vascular sprouting in collagen with Matrigel lattices. PAI-1(-/-) vessels showed an increased capillary sprouting in both collagen and Matrigel. Deficiency of uPA significantly reduced the rate of sprouting, whereas tPA(-/-) vessels showed a profound inhibition of capillary sprouting. The Plg(-/-) vessels failed to sprout, a defect that was restored not only by exogenous Plg, but also by the addition of PAs; a nonproteolytic effect of tPA was observed in Matrigel. Zymography revealed no differences in the activity of metalloproteinase (MMP)-2 and -9 in wild-type and PAI-1(-/-) vessels, but demonstrated reduced MMP-9 activity in all angiogenesis-deficient vessels. In summary, 1) PAI-1 by itself is a modest inhibitor of endothelial sprouting, 2) tPA and Plg are indispensable for angiogenesis in this model, 3) Plg is not the only substrate for PAs, and 4) the activity of MMP-9 is undetectable in explant cultures from tPA and Plg knockout mice.     

High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and alpha(v)beta(3)-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.     

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.     

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate that beta1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting beta1 integrins may effectively impair neovascularization.     

PECAM-1 isoform-specific regulation of kidney endothelial cell migration and capillary morphogenesis

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in angiogenesis through its involvement in endothelial cell-cell and cell-matrix interactions and signal transduction. Recent studies indicate that the cytoplasmic domain of PECAM-1 plays an important role in its cell adhesive and signaling properties. However, the role PECAM-1 isoforms play during angiogenic events such as cell adhesion and migration requires further delineation. To gain insight into the role PECAM-1 plays during vascular development and angiogenesis, we examined the expression pattern of PECAM-1 isoforms during kidney vascularization. We show that multiple isoforms of PECAM-1 are expressed during renal vascular development with different frequencies. The PECAM-1 that lacks exons 14 and 15 (14&15) was the predominant isoform detected in the renal vasculature. To further study PECAM-1 isoform-specific functions we isolated kidney endothelial cells (EC) from wild-type and PECAM-1-deficient (PECAM-1–/–) mice with B₄-lectin-coated magnetic beads. PECAM-1–/– kidney EC showed reduced migration, inability to undergo capillary morphogenesis in Matrigel, dense peripheral focal adhesions, and peripheral cortical actin distribution compared with wild-type cells. PECAM-1–/– kidney EC secreted increased amounts of fibronectin and decreased amounts of tenascin-C and thrombospondin-1. Reexpression of 14&15, but not full-length, PECAM-1 in PECAM-1–/– kidney EC restored cell migration and capillary morphogenesis defects. Thus PECAM-1 may regulate the adhesive and migratory properties of kidney EC in an isoform-specific fashion through modulation of integrin activity and extracellular matrix protein expression. Our results indicate that regulated expression of specific PECAM-1 isoforms may enable EC to accommodate the different stages of angiogenesis. CD31; alternative splicing; angiogenesis; integrins; extracellular matrix     

Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement

Angiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic 'cell-mixing'. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the 'cell-mixing' phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via γ-secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.     

Cep70 contributes to angiogenesis by modulating microtubule rearrangement and stimulating cell polarization and migration

Centrosomal proteins intricately regulate diverse microtubule-mediated cellular activities, including cell polarization and migration. However, the direct participation of these proteins in angiogenesis, which involves vascular endothelial cell migration from preexisting blood vessels, remains elusive. Here we show that the centrosomal protein Cep70 is necessary for angiogenic response in mice. This protein is also required for tube formation and capillary sprouting in vitro from vascular endothelial cells. Wound healing and transwell assays reveal that Cep70 plays a significant role in endothelial cell migration. Depletion of Cep70 results in severe defects in membrane ruffling and centrosome reorientation, indicating a requirement for this protein in cell polarization. In addition, Cep70 is critically involved in microtubule rearrangement in response to the migratory stimulus. Our data further demonstrate that Cep70 is important for Cdc42 and Rac1 activation to promote angiogenesis. These findings thus establish Cep70 as a crucial regulator of the angiogenic process and emphasize the significance of microtubule rearrangement and cell polarization and migration in angiogenesis.     

Thrombospondin-1 deficient mice exhibit an altered expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature and endothelial cells

We have previously shown that thrombosponsin-1 (TSP1) and PECAM-1 are components of a regulatory switch whose reciprocal regulation in the endothelial cells (EC) promotes an angiogenic or a differentiated, quiescent phenotype. The physiological role TSP1 plays in modulation of PECAM-1 expression and function during vascular development and angiogenesis remains largely unknown. Here we demonstrate that PECAM-1 undergoes alternative splicing in its cytoplasmic domain generating eight isoforms in the retinal vasculature of wild type and TSP1-/- mice. All PECAM-1 isoforms examined contained exon 13. The frequency of PECAM-1 isoform(s) containing exon 14 was significantly higher during early stages of retinal vascularization, which decreased during later stages of retinal vascularization in wild type mice. In contrast, the frequency of exon 14 containing PECAM-1 isoform(s) did not significantly change during retinal vascularization in TSP1-/- mice. They consistently expressed higher number of isoforms with exon 14 during later stages of retinal vascularization. The higher level of PECAM-1 isoforms with exon 14 was also observed in cultured TSP1-/- retinal EC compared to wild type retinal EC. This was consistent with increased amounts of Src and SHP-2 associated with PECAM-1, and enhanced migration and proliferation in TSP1-/- retinal EC. These data suggest PECAM-1 signaling in the endothelium is modulated by its alternative splicing during retinal vascular development and angiogenesis, which may be impacted by TSP1 expression.     

A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice

Interactions between astrocytes and endothelial cells (ECs) are crucial for retinal vascular formation. Astrocytes induce migration and proliferation of ECs via their production of vascular endothelial growth factor (VEGF) and, conversely, ECs induce maturation of astrocytes possibly by the secretion of leukemia inhibitory factor (LIF). Together with the maturation of astrocytes, this finalizes angiogenesis. Thus far, the mechanisms triggering LIF production in ECs are unclear. Here we show that apelin, a ligand for the endothelial receptor APJ, induces maturation of astrocytes mediated by the production of LIF from ECs. APJ (Aplnr)- and Apln-deficient mice show delayed angiogenesis; however, aberrant overgrowth of endothelial networks with immature astrocyte overgrowth was induced. When ECs were stimulated with apelin, LIF expression was upregulated and intraocular injection of LIF into APJ-deficient mice suppressed EC and astrocyte overgrowth. These data suggest an involvement of apelin/APJ in the maturation process of retinal angiogenesis.     

In vitro and in vivo antiangiogenic properties of the serpin protease nexin-1

The serpin protease nexin-1 (PN-1) is expressed by vascular cells and secreted by platelets upon activation, and it is known to interact with several modulators of angiogenesis, such as proteases, matrix proteins, and glycosaminoglycans. We therefore investigated the impact of PN-1 on endothelial cell angiogenic responses in vitro and ex vivo and in vivo in PN-1-deficient mice. We found that PN-1 is antiangiogenic in vitro: it inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell responses, including proliferation, migration, and capillary tube formation, and decreased cell spreading on vitronectin. These effects do not require the antiprotease activity of PN-1 but involve PN-1 binding to glycosaminoglycans. In addition, our results indicated that PN-1 does not act by blocking VEGF binding to its heparan sulfate proteoglycan coreceptors. The results obtained in vitro were supported ex vivo in PN-1-deficient mice, where the microvascular network sprouting from aortic rings was significantly enhanced. Moreover, in vivo, neovessel formation was promoted in the Matrigel plug assay in PN-1-deficient mice compared to wild-type mice, and these effects were reversed by the addition of recombinant PN-1. Taken together, our results demonstrate that PN-1 has direct antiangiogenic properties and is a yet-unrecognized player in the angiogenic balance.     

Attenuation of retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy in Bcl-2-/- mice

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, we investigated the physiological role of bcl-2 in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibited a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. We observed, in bcl-2-/- mice, delayed development of retinal vasculature and remodeling, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibited a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization was significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR.