Development and pathology of the hyaloid, choroidal and retinal vasculature

During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.     

Angiopoietin-2 plays an important role in retinal angiogenesis

Angiopoietin 2 (Ang2) expression in the retina is increased during physiologic and pathologic neovascularization suggesting that it may be involved. In this study, we used Ang2-deficient mice to test that hypothesis. Mice deficient in Ang2 showed delayed and incomplete development of the superficial vascular bed of the retina, which develops primarily by vasculogenesis, and complete absence of the intermediate and deep vascular beds which develop by angiogenesis. In addition to incomplete retinal vascular development, Ang2-deficient mice showed lack of regression of the hyaloid vasculature, resulting in a phenotype that mimics infants with persistent fetal vasculature (PFV), a relatively common congenital abnormality. Exposure to high levels of oxygen resulted in partial regression of the retinal vessels, indicating that oxygen-induced regression of retinal vessels does not require Ang2. When these oxygen-exposed mice with few retinal vessels were moved to room air, there was no ischemia-induced retinal neovascularization. These data support the hypothesis that Ang2 plays a critical role in physiologic and pathologic angiogenesis, and physiologic, but not oxygen-induced vascular regression. The data also suggest that infants with PFV should be examined for genetic modifications that would be expected to cause perturbations in Tie2 signaling.     

Birdshot retinochoroidopathy

Birdshot retinochoroidopathy is a rare bilateral ocular disorder which typically affects healthy, middle-age caucasians. The typical symptoms are recent onset of bilateral floaters with decreased vision in a white and quiet eye. Minimal anterior chamber reaction, vitreous infiltration without snowballs, and multifocal depigmented lesions in the outer retina and choroid are characteristic of birdshot retinochoroidopathy. Other associated findings include retinal vasculitis, paravascular hemorrhage, vitreous hemorrhage, cystoid macular edema, cellophane maculopathy, posterior subcapsular cataract, optic disc edema, vascular attenuation and tortuosity, optic atrophy and retinal or choroidal neovascularization. Management involves Amsler grid self-assessment and routine funduscopic examinations. A recently documented case of treatment with aromatic retinoids showed improvement while many other commonly prescribed medications are equivocal in effect. Laser photoablation or panretinal photocoagulation are recommended for treatable choroidal or retinal neovascularization, respectively.     

Prolonged blockade of VEGF family members does not cause identifiable damage to retinal neurons or vessels

Several ocular diseases complicated by neovascularization are being treated by repeated intraocular injections of vascular endothelial growth factor (VEGF) antagonists. While substantial benefits have been documented, there is concern that unrecognized damage may be occurring, because blockade of VEGF may damage the fenestrated vessels of the choroicapillaris and deprive retinal neurons of input from a survival factor. One report has suggested that even temporary blockade of all isoforms of VEGF-A results in significant loss of retinal ganglion cells. In this study, we utilized double transgenic mice with doxycycline-inducible expression of soluble VEGF receptor 1 coupled to an Fc fragment (sVEGFR1Fc), a potent antagonist of several VEGF family members, including VEGF-A, to test the effects of VEGF blockade in the retina. Expression of sVEGFR1Fc completely blocked VEGF-induced retinal vascular permeability and significantly suppressed the development of choroidal neovascularization at rupture sites in Bruch's membrane, but did not cause regression of established choroidal neovascularization. Mice with constant expression of sVEGFR1Fc in the retina for 7 months had normal electroretinograms and normal retinal and choroidal ultrastructure including normal fenestrations in the choroicapillaris. They also showed no significant difference from control mice in the number of ganglion cell axons in optic nerve cross sections and the retinal level of mRNA for 3 ganglion cell-specific genes. These data indicate that constant blockade of VEGF for up to 7 months has no identifiable deleterious effects on the retina or choroid and support the use of VEGF antagonists in the treatment of retinal diseases.     

Cell injury unmasks a latent proangiogenic phenotype in mice with increased expression of FGF2 in the retina

Fibroblast growth factor-2 (FGF2) is a potent mitogen for vascular endothelial cells and exogenous administration of FGF2 stimulates angiogenesis. However, increased expression of FGF2 in the retina does not cause angiogenesis. One possible explanation is that FGF2 may not be capable of initiating angiogenesis unless it is administered in pharmacologic levels or there is coexpression of another angiogenic factor. Alternatively, there may be control mechanisms that sequester FGF2 in vivo, preventing it from manifesting its in vitro angiogenic activity. We tested the first hypothesis by crossing mice that express FGF2 in the retina with mice that express vascular endothelial growth factor (VEGF) in the retina. Surprisingly, despite comparable levels of VEGF expression, mice that expressed both FGF2 and VEGF had significantly less neovascularization than mice that expressed VEGF alone. The second hypothesis was tested by treating Rho/FGF2 transgenic mice with low-intensity laser photocoagulation that disrupts photoreceptors, but does not rupture Bruch's membrane, or intense laser that ruptures Bruch's membrane. In Rho/FGF2 transgenics, but not wild type mice, choroidal neovascularization developed in areas of low-intensity laser. Both wild type and transgenic mice developed choroidal neovascularization in areas of intense laser that ruptured Bruch's membrane, but the area of neovascularization was significantly greater in transgenics. These data suggest that increased retinal expression of FGF2 is angiogenic only when it is accompanied by cell injury that overcomes sequestration control mechanisms.     

Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization.

In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.     

Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy

Abstract

The vasculature forms a highly branched network investing every organ of vertebrate organisms. The retinal circulation, in particular, is supported by a central retinal artery branching into superficial arteries, which dive into the retina to form a dense network of capillaries in the deeper retinal layers. The function of the retina is highly dependent on the integrity and proper functioning of its vascular network and numerous ocular diseases including diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity are caused by vascular abnormalities culminating in total and sometimes irreversible loss of vision. CCN1 and CCN2 are inducible extracellular matrix (ECM) proteins which play a major role in normal and aberrant formation of blood vessels as their expression is associated with developmental and pathological angiogenesis. Both CCN1 and CCN2 achieve disparate cell-type and context-dependent activities through modulation of the angiogenic and synthetic phenotype of vascular and mesenchymal cells respectively. At the molecular level, CCN1 and CCN2 may control capillary growth and vascular cell differentiation by altering the composition or function of the constitutive ECM proteins, potentiating or interfering with the activity of various ligands and/or their receptors, physically interfering with the ECM-cell surface interconnections, and/or reprogramming gene expression driving cells toward new phenotypes. As such, these proteins emerged as important prognostic markers and potential therapeutic targets in neovascular and fibrovascular diseases of the eye. The purpose of this review is to highlight our current knowledge and understanding of the most recent data linking CCN1 and CCN2 signaling to ocular neovascularization bolstering the potential value of targeting these proteins in a therapeutic context.     

Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization.

High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the development of subretinal choroidal angiogenesis induced by laser photocoagulation. When systemic and local PAI-1 expression was achieved by intravenous injection of a replication-defective adenoviral vector expressing human PAI-1 cDNA, the wild-type pattern of choroidal angiogenesis was restored. These observations demonstrate the proangiogenic activity of PAI-1 not only in tumoral models, but also in choroidal experimental neovascularization sharing similarities with human AMD. They identify therefore PAI-1 as a potential target for therapeutic ocular anti-angiogenic strategies.     

Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.     

Blockade of sphingosine-1-phosphate reduces macrophage influx and retinal and choroidal neovascularization

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that stimulates endothelial cell migration, proliferation, and survival in vitro, and tumor angiogenesis in vivo. In this study, we used a humanized monoclonal antibody (sonepcizumab) that selectively binds S1P to investigate its role in retinal and choroidal neovascularization (NV). Intraocular injection of sonepcizumab significantly reduced macrophage influx into ischemic retina and strongly suppressed retinal NV in mice with oxygen-induced ischemic retinopathy. In mice with laser-induced rupture sites in Bruch's membrane, intraocular injection of sonepcizumab significantly reduced the area of choroidal NV and concomitantly reduced fluorescein leakage from the remaining choroidal NV. Four weeks after intraocular injection of up to 1.8 mg of the sonepcizumab in non-human primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV.     

Bim is responsible for the inherent sensitivity of the developing retinal vasculature to hyperoxia

Apoptosis plays an important role in development and remodeling of vasculature during organogenesis. Coordinated branching and remodeling of the retinal vascular tree is essential for normal retinal function. Bcl-2 family members, such as bim not only influence apoptosis, but also cell adhesive and migratory properties essential during vascular development. Here we examined the impact of bim deficiency on postnatal retinal vascularization, as well as retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) and laser-induced choroidal neovascularization. Loss of bim expression was associated with increased retinal vascular density in mature animals. This was mainly attributed to increased numbers of pericytes and endothelial cells. However, the initial spread of the superficial layer of retinal vasculature and, the appearance and density of the tip cells were similar in bim+/+ and bim−/− mice. In addition, hyaloid vessel regression was attenuated in the absence of bim. Furthermore, in the absence of bim retinal vessel obliteration and neovascularization did not occur during OIR. Instead, normal inner retinal vascularization proceeded independent of changes in oxygen levels. In contrast, choroidal neovascularization occurred equally well in bim+/+ and bim−/− mice. Together our data suggest bim expression may be responsible for the inherent sensitivity of the developing retinal vasculature to changes in oxygen levels, and promotes vessel obliteration in response to hyperoxia.     

Notch signaling in ocular vasculature development and diseases

Ocular angiogenesis, characterized by the formation of new blood vessels in the avascular area in eyes, is a highly coordinated process involved in retinal vasculature formation and several ocular diseases such as age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. This process is orchestrated by complicated cellular interactions and vascular growth factors, during which endothelial cells acquire heterogeneous phenotypes and distinct cellular destinations. To date, while the vascular endothelial growth factor has been identified as the most critical angiogenic agent with a remarkable therapeutic value, the Notch signaling pathway appears to be a similarly important regulator in several angiogenic steps. Recent progress has highlighted the involvement, mechanisms and therapeutic potential of Notch signaling in retinal vasculature development and pathological angiogenesis-related eye disorders, which may cause irreversible blindness.     

Seeing the light: new insights into the molecular pathogenesis of retinal diseases

In the past, most treatments for retinal diseases have been empirical. Steroids and/or laser photocoagulation and/or surgery have been tried for almost every condition with little or no understanding of the underlying disease. Over the past several years vision researchers have uncovered molecular components of processes, such as visual transduction and the visual cycle, that are critical for visual function, and identified other molecules that lead to dysfunction and disease processes such as neovascularization and macular edema. It is becoming clear that dysregulation of certain molecules can have major effects on retinal structure and function. Studies in animal models have suggested that inhibiting or augmenting levels of a single molecule can have major effects in complex disease processes. Although several molecules probably contribute to neovascularization and excessive vascular permeability in the eye, blockade of vascular endothelial growth factor (VEGF) has remarkable beneficial effects in animal models that have now been proven to apply to human diseases in clinical trials. Intraocular injection of VEGF antagonists has revolutionized the treatment of choroidal neovascularization (CNV) and macular edema and serves as a model of targeted ocular pharmacotherapy. Significant progress elucidating the molecular pathogenesis of several disease processes in the eye may soon lead to new treatments following the lead of VEGF antagonists. Initial treatments that provide benefit from frequent intraocular injections are likely to be followed by sustained delivery of drugs and/or prolonged protein delivery by gene transfer. The eye has entered the era of molecular therapy.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

Nitric oxide is proangiogenic in the retina and choroid

Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. In this study, we used mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) to investigate the effects of NO in ocular neovascularization. In transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of inducible NOS (iNOS) or neuronal NOS (nNOS), but not endothelial NOS (eNOS), caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization and that different isoforms of NOS are involved in different settings and different disease processes. A broad spectrum NOS inhibitor may have therapeutic potential for treatment of both retinal and choroidal neovascularization.     

Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema

Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.     

Therapeutic efficacy of Stephania tetrandra S. Moore for treatment of neovascularization of retinal capillary (retinopathy) in diabetes--in vitro study.

The present study was designed to examine therapeutic efficacy of the root extract of Stephania Tetrandra S. Moore (STMS) (traditional Chinese medicine; Han Fang Ji) for treatment of neovascularization of the retinal capillary (retinopathy) in streptozotocin (STZ)-induced diabetic rats (STZ diabetic rats) in culture. Recently we have established the culture system in which fetal bovine serum (FBS) in Dulbecco modified Eagle medium (DMEM) induced neovascularization of the retinal capillary and choroidal capillary in normal rats in culture. STZ diabetic rats showed more neovascularization of the retinal capillary and choroidal capillary than did normal rats in culture. In this study, the retinal tissue was removed for the posterior ocular region and cultured in DMEM containing FBS. The choroidal tissue of the posterior ocular region was also removed and cultured as an internal reference. Administration of STSM (0.91, 9.1 and 91 microg/ml) significantly suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary; administration of STSM suppressed neovascularization of the choroidal capillary in both STZ diabetic rats and normal rats. In order to determine the component of STSM inhibiting neovascularization of the retinal capillary, tetrandrine (a major chemical constituent of STSM) was administered and neovascularization of the retinal capillary was examined in culture. The effect of tetrandrine on the choroidal capillary was also examined as an internal reference. Administration of tetrandrine (0.1, 1.0 and 10 microM) suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary of both STZ diabetic rats and normal rats. We infer, therefore, that STSM has a direct effect on the retinal capillary of posterior ocular region and suppresses neovascularization of retinal capillary in STZ diabetic rats through the activation of tetrandrine. These results suggest that STSM may prevent for delay the progression of retinopathy in diabetic patients.     

Hypoxia-induced insulin-like growth factor II contributes to retinal vascularization in ocular development

In ocular development, retinal physiological hypoxia in response to the retinal metabolic activity controls retinal vascular development, which is regulated by variable angiogenic factors. Herein, we demonstrated that hypoxia-induced IGF-II could contribute to retinal vascularization in ocular development. In the developing retina, IGF-II expression appears to be predominant on retinal vessels, which was chronologically increased and peaked during active retinal angiogenesis similar to VEGF expression. Under hypoxic condition, IGF-II as well as VEGF was significantly up-regulated in retinal vascular endothelial cells. In addition, IGF-II treatment could also increase VEGF expression in retinal vascular endothelial cells. The VEGF expression induced by IGF-II was mediated by ERK-1/2 activation. Moreover, IGF-II strongly promoted angiogenic processes of migration and tube formation of retinal microvascular endothelial cells. In conclusion, our results provided that hypoxia-induced IGF-II may regulate retinal vascular development not only directly by IGF-II-mediated angiogenic activity, but also indirectly by IGF-II-induced VEGF expression. Therefore, the potential contribution of IGF-II to pathological retinal angiogenesis should be furthermore explored for the development of novel treatments to vaso-proliferative retinopathies.     

Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis

Adult bone marrow (BM) contains cells capable of differentiating along hematopoietic (Lin(+)) or non-hematopoietic (Lin(-)) lineages. Lin(-) hematopoietic stem cells (HSCs) have recently been shown to contain a population of endothelial precursor cells (EPCs) capable of forming blood vessels. Here we show that intravitreally injected Lin(-) BM cells selectively target retinal astrocytes, cells that serve as a template for both developmental and injury-associated retinal angiogenesis. When Lin(-) BM cells were injected into neonatal mouse eyes, they extensively and stably incorporated into forming retinal vasculature. When EPC-enriched HSCs were injected into the eyes of neonatal rd/rd mice, whose vasculature ordinarily degenerates with age, they rescued and maintained a normal vasculature. In contrast, normal retinal angiogenesis was inhibited when EPCs expressing a potent angiostatic protein were injected. We have demonstrated that Lin(-) BM cells and astrocytes specifically interact with one another during normal angiogenesis and pathological vascular degeneration in the retina. Selective targeting with Lin(-) HSC may be a useful therapeutic approach for the treatment of many ocular diseases.     

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.