Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Therapeutic antibodies targeting angiomotin inhibit angiogenesis in vivo.

We have previously shown that angiomotin (Amot) mediates angiostatin inhibition of endothelial migration and tube formation in vitro. A crucial role of angiomotin in regulating endothelial cell motility is indicated by the findings that knockdown of Amot in zebrafish reduces the number of filopodia of endothelial tip cells and severely impairs the migration of intersegmental vessels. In addition, targeting angiomotin using DNA vaccination inhibits angiogenesis and tumor growth in vivo. In this report, we have generated antibodies that, similar to angiostatin, bind to angiomotin on the endothelial cell surface. These antibodies inhibited FGF-2 and vascular endothelial growth factor (VEGF) -induced endothelial migration in the Boyden chamber assay. Furthermore, the anti-Amot B06 antibody significantly reduced the number of endothelial filopodia and inhibited vessel migration during retinal angiogenesis in vivo. We also show that systemic or local treatment with this antibody inhibits pathological blood vessel formation associated with tumor growth or laser-induced choroid neovascularization of the eye. These findings provide a rationale for using angiomotin antibodies for specifically targeting endothelial migration in angiogenesis-dependent diseases.

Protective role of reactive astrocytes in brain ischemia.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

Growth factor gradients in vascular patterning.

Growth factor gradients regulate many developmental processes. VEGF-A is distributed in a graded fashion in growing tissues in order to direct sprouting of new vessels. Growth factor gradients can be formed by regulated production, retention, controlled release and degradation. VEGF-A production is controlled by hypoxia while its retention depends on the C-terminal heparin-binding motifs present in the longer splice-isoforms, VEGF164 and 188. This motif confers binding to the cell surface and the surrounding extracelluar matrix. The short isoform VEGF120 is diffusible and hence fails to direct endothelial tip cell migration. Conditional inactivation of heparan sulfate proteoglycans in the cells that produce VEGF results similarly in misguidance of the tip cells. Studying retinal developmental angiogenesis and pathological neovascularization side-by-side in the mouse retina, we find that endothelial tip cell guidance and stalk cell proliferation control are disrupted in neovascularization due to a loss of VEGF-A retention. The cause for this is proteolytic cleavage of VEGF-A by matrix metalloproteases (MMP) derived mostly from macrophages infiltrating the ischaemic retinal areas. Genetic or pharmacological inhibition of macrophage infiltration or MMP activity can rescue guided revascularization at the expense of pre-retinal neovascularization. Disruption of VEGF-A gradients provides a novel concept for the mechanism underlying pathological patterning in ocular disease.

Under stress, the absence of intermediate filaments from Müller cells in the retina has structural and functional consequences.

In epithelial and muscle cells, intermediate filaments (IFs) are important for resistance to mechanical stress. The aim of this study was to elucidate whether IFs are also important for providing resistance to mechanical stress in the Müller cells of the retina and whether this has any pathophysiological consequences. We used mice deficient in IF proteins glial fibrillary acidic protein and/or vimentin (GFAP(-/-), Vim(-/-) and GFAP(-/-) Vim(-/-)), and stress on the retina was applied by excision of the eyes immediately post mortem (compared with in situ fixation) or by inducing a neovascular response to oxygen-induced retinopathy (OIR). The structure of unchallenged retinas was normal, but mechanical stress caused local separation of the inner limiting membrane (ILM) and adjacent tissue from the rest of the retina in GFAP(-/-) Vim(-/-) mice and, to a lesser extent, in Vim(-/-) mice. This detachment occurred within the endfeet of Müller cells, structures normally rich in IFs but IF-free in GFAP(-/-) Vim(-/-) mice. Hypoxia-induced neovascularization was comparable in all groups of mice with respect to the retinal surface area occupied by new vessels. However, the vessels traversed the ILM and penetrated the vitreous body less frequently than in wild-type retinas (31-55% in Vim(-/-), 66-79% in GFAP(-/-) Vim(-/-)). We conclude that IFs are important for maintaining the mechanical integrity of Müller-cell endfeet and the inner retinal layers under a mechanical challenge. Furthermore, the absence of IFs in Müller cells leads to an abnormal response of the vascular system to ischemia, specifically decreased ability of newly formed blood vessels to traverse the ILM.

Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin.

With recent progress in neuroscience and stem-cell research, neural transplantation has emerged as a promising therapy for treating CNS diseases. The success of transplantation has been limited, however, by the restricted ability of neural implants to survive and establish neuronal connections with the host. Little is known about the mechanisms responsible for this failure. Neural implantation triggers reactive gliosis, a process accompanied by upregulation of intermediate filaments in astrocytes and formation of astroglial scar tissue. Here we show that the retinas of adult mice deficient in glial fibrillary acidic protein and vimentin, and consequently lacking intermediate filaments in reactive astrocytes and Müller cells, provide a permissive environment for grafted neurons to migrate and extend neurites. The transplanted cells integrated robustly into the host retina with distinct neuronal identity and appropriate neuronal projections. Our results indicate an essential role for reactive astroglial cells in preventing neural graft integration after transplantation.

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.

A new method for large scale isolation of kidney glomeruli from mice.

Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5- micro m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 +/- 4699 (mean +/- SD, n = 14) with a purity of 97.5 +/- 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.