David T. Shima

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.

Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Pegaptanib sodium (Macugen; Eyetech Pharmaceuticals/Pfizer) is an RNA aptamer directed against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for pathological ocular neovascularization and vascular permeability. After nearly a decade of preclinical development to optimize and characterize its biological effects, pegaptanib was shown in clinical trials to be effective in treating choroidal neovascularization associated with age-related macular degeneration. Pegaptanib therefore has the notable distinction of being the first aptamer therapeutic approved for use in humans, paving the way for future aptamer applications.

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF 164 and VEGF 188

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability

Vessel growth is often associated with ischemia. VEGF, a potent angiogenic factor, has been shown to be induced by low oxygen concentrations. These studies were conducted to investigate the molecular basis of the hypoxia‐induced increase in VEGF mRNA. Run‐on analysis of VEGF revealed a minimal increase in the rate of gene transcription in a human retinal epithelial cell line grown under hypoxic conditions. Examination of VEGF mRNA stability revealed that the half‐life of VEGF transcripts under normoxia was short, 30–45 min, but was dramatically increased to 6–8 h in cells grown under hypoxia. Cobalt chloride, which elevates VEGF and has been suggested to be similar to hypoxia in its mechanism of action, had only a slight effect on decay rate. We postulate that hypoxia‐induced increases in mRNA stability provide the sustained increases in VEGF mRNA levels necessary to support a neovascular response.

The role of vascular endothelial growth factor in ocular health and disease.

The leading causes of blindness are retinal and choroidal diseases manifesting abnormal vessel permeability and growth. Scientists have sought to understand the mechanisms underlying these pathologic conditions with the hope of developing directed and effective pharmacologic therapies. Research has yielded important new mechanistic data, making possible the development of new drugs. One of the most important targets to have emerged is a secreted protein named vascular endothelial growth factor. This review will summarize the current state of our knowledge regarding this growth factor and outline some important questions that remain to be answered.

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE‐cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE‐PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor‐type phosphatase, co‐precipitates with VE‐cadherin, but not with β‐catenin, from cell lysates of transfected COS‐7 cells and of endothelial cells. Co‐precipitation of VE‐cadherin and VE‐PTP required the most membrane‐proximal extracellular domains of each protein. Expression of VE‐PTP in triple‐transfected COS‐7 cells and in CHO cells reversed the tyrosine phosphorylation of VE‐cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR‐2). Expression of VE‐PTP under an inducible promotor in CHO cells transfected with VE‐cadherin and VEGFR‐2 increased the VE‐cadherin‐mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE‐PTP had the same effect on VE‐cadherin phosphorylation and cell layer permeability. Thus, VE‐PTP is a transmembrane binding partner of VE‐cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE‐cadherin and cell layer permeability independently of its enzymatic activity.

Neuropilin-1 is required for endothelial tip cell guidance in the developing central nervous system.

Recent evidence indicates that sprouting angiogenesis in the central nervous system (CNS) is a guided process similar to the guidance of axons and insect tracheal tubes. Specialized tip cells of vessel sprouts navigate in response to local depots or gradients of vascular endothelial growth factor (VEGF‐A). Neuropilin‐1 (Nrp‐1) is a transmembrane receptor with a repulsive function in axon guidance. Nrp‐1 also binds the VEGF‐A splice isoform VEGF165, stimulates angiogenesis, and is necessary for vascular development in the mouse. However, the morphogenetic events controlled by Nrp‐1 in angiogenesis have not been defined. Here, we analyzed endothelial tip cell guidance in the CNS of Nrp‐1–deficient mice. We focused our attention on the developing hindbrain, which is normally vascularized in a stereotyped manner. Initially, angiogenic sprouts extend along radial glia from the pial surface toward the ventricles, but in the subventricular zone (SVZ), they leave the radial path, turn laterally, and fuse to form a capillary plexus. Radial sprout elongation correlated with tip cell filopodia extensions along nestin‐positive radial glial processes, but in the SVZ, the tip cell filopodia also extended perpendicular to the glial tracks and made contact with filopodia of the neighboring sprouts. In Nrp‐1–deficient mice, the tip cell filopodia remained associated with the radial glia in the SVZ, which correlated with a failure of sprout turning and elongation across this region. As a result, the sprouts remained blind‐ended forming glomeruloid tufts in the SVZ. These observations suggest that Nrp‐1 plays an important role in allowing the endothelial tip cell filopodia to switch substrate and protrude in a new direction at a specific location in the developing brain. Developmental Dynamics 231:503–509, 2004.

In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.

To investigate the in vivo angiogenic activity of placenta growth factor (PIGF) and its heterodimers with vascular endothelial growth factor (VEGF), the induction of neovascularization of these factors in the mouse cornea was studied. VEGF165 is sufficiently potent to stimulate new capillary growth from the limbal vessels. PIGF129/VEGF165 heterodimers also induce corneal neovascularization with a maximal vessel length similar to VEGF165, but with a marked decrease of vessel density. In contrast, PIGF129 has little or no effect in this in vivo angiogenesis assay. The expression of VEGF mRNA and protein is drastically up-regulated by hypoxia in choriocarcinoma cells, whereas expression of PIGF is not affected by the low concentration of oxygen. Up-regulation of VEGF production results in increased formation of PIGF/VEGF heterodimers in these tumor cells. Thus, hypoxia indirectly up-regulates expression levels of PIGF/VEGF heterodimers and modulates VEGF activity when these factors are co-expressed.

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589–601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP–Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.