Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch.

Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome.

Egfl7 is differentially expressed in arteries and veins during retinal vascular development.

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRß blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Topotecan hydrochloride effects on retinal vessels in newborn rats.

A physiological system, i.e. rodent retina during vessel formation and hierarchical organization, was utilised for assaying antiangiogenic properties of Topotecan, a topoisomerase I inhibitor, capable of inhibiting tumoral growth in animal models of retinoblastoma. In particular we analysed possible differences in effectiveness and side effects among different drug dosages and ways of administration. In the present research only qualitative analyses were undertaken. After preliminary experiments, in which suckling animals subcutaneously treated with Topotecan dosages comprised between 9 and 3 mg/kg underwent high lethality and extremely severe systemic damages, 7 day-old rats were subcutaneously, intravenously or peribulbary injected with a single dose of 1 mg/kg; retinal vessels were visualized in retinal fluorangio-graphies taken 1 and 2 weeks after treatment. The most important and frequent alterations were found to affect radial vessels, which showed non-perfused and/or regionally mislocated segments, together with abnormal branching and enlargements in retinal periphery; persistence of capillary-free periarteriolar regions, non-vascularised regions and spots of extravascular FITC were also detected. Despite the high individual variability the alterations were substantially similar among the different ways of drug administration, while they appeared milder in 21 day-old rats, with respect to younger ones. The extensive vascular remodelling found after Topotecan administration, besides demonstrating the antiangiogenic properties of this chemioterapic drug, confirms the rodent retina as a highly valuable model system for studying angiogenesis modulation.

Smaller birth size is associated with narrower retinal arterioles in early adolescence.

OBJECTIVE: In the current study, we aimed to examine the associations of low birth weight with retinal vascular caliber and vascular fractal dimension during early adolescence. METHODS: A population-based study of 12-year-old schoolchildren (2353/3144 [75.3%]) recruited from a random cluster sample of 21 schools. Birth weight, birth length and head circumference were obtained via parent report of the child's birth record. Retinal images were taken and vessel diameter and fractal dimension were quantified using validated computer-based methods. RESULTS: After adjusting for age, sex, ethnicity, body mass index, iris color, axial length, mean arterial blood pressure, prematurity and fellow retinal vascular caliber, children in the lowest quartiles of birth weight had ∼2.5 µm narrower mean retinal arteriolar caliber than those in the highest quartiles (p for trend = 0.001). Associations were observed between shorter birth length and smaller head circumference with narrower retinal arterioles. Smaller head circumference was associated with decreased fractal dimension (p for trend = 0.03). CONCLUSIONS: Children with lower birth weight were more likely to have narrower retinal arterioles, while those with smaller head circumference were more likely to have reduced complexity of their retinal microvasculature. These variations in microvascular structure in adolescence could reflect a susceptibility to cardiovascular disease during adulthood, resulting from a disadvantaged growth environment in utero.

Expression of ATP-binding cassette transporters at the inner blood-retinal barrier in a neonatal mouse model of oxygen-induced retinopathy.

ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB) are responsible for the majority of the transcellular movement of various substrates, including various drugs, and contribute to the maintenance of brain homeostasis. Clinically, the abnormal expression of efflux transporters at the BBB is known to be associated with brain diseases such as epilepsy. In the retina, vascular endothelial cells outline the inner blood-retinal barrier (BRB) like the BBB, and some ABC efflux transporters are expressed in the adult retina. However, little is known about ABC transporter expression during retinal development or under pathological conditions. Here, we examined ABC transporter expression in the mouse retina, and demonstrated that P-glycoprotein (P-gp)/ABCB1, Mrp4/ABCC4, and Bcrp/ABCG2 were almost uniformly expressed in these blood vessels, including the capillaries and large vessels. This expression persisted throughout the developmental period, and the hyaloid vessels that normally feed the developing eye were immunoreactive for P-gp and Mrp4. Furthermore, we investigated ABC transporter expression in pathological angiogenesis using an oxygen-induced retinopathy model where hypoxia-induced preretinal neovascularization occurred around the central avascular retina. P-gp was prominently immunoexpressed but Mrp4 and Bcrp were weakly immunoexpressed, in the preretinal neovascular tufts. These findings will be helpful for understanding the roles of ABC transporters during both physiological and pathological retinal angiogenesis, and might provide new insights for safe and effective drug administration to infants or patients with angiogenic ocular disease.

Contextual role for angiopoietins and TGFbeta1 in blood vessel stabilization.

We used a 3D in-vitro model of angiogenesis to investigate the effects of different growth factors on vessel formation and stabilization in vitro. Vascular endothelial growth factor (VEGF) was the only factor that induced the formation, elongation and sprouting of capillary-like structures (CLS) by bovine retinal capillary endothelial cells (BREC), an effect that was dose-dependent and saturable. Basic fibroblast growth factor 2 (FGF2) enhanced capillary formation in the presence of VEGF, leading to a more complex network of CLS and a higher rate of BrdU incorporation than VEGF alone, indicating that whereas VEGF acts as a morphogen, FGF2 is primarily a mitogen. Addition of transforming growth factor beta1 (TGFbeta1) to the 3D assay along with VEGF and FGF2, reduced tube formation in a dose-dependent manner. When added at the time of cell plating TGFbeta1 completely suppressed formation of VEGF/FGF2-stimulated CLS. Angiopoietin 1 (Ang1) prevented regression of the TGFbeta1-induced CLS, an effect that was blocked by angiopoietin 2 (Ang2), but required the continuous presence of VEGF.

Regulation of GDNF and its receptor components GFR-alpha1, -alpha2 and Ret during development and in the mature retino-collicular pathway.

The development of the retino-tectal projection as part of the central visual pathway is accomplished around postnatal day (P) 10-14 in rodents, and trophic factors are important for topographic refinement of this projection. Emerging data indicate that GDNF may influence synaptic plasticity of this projection. To date, maturation-dependent kinetics of GDNF release and expression and biological function of single GDNF receptors along the retino-collicular pathway are ill-defined. Here, we examined mRNA and protein expression of GDNF and its multicomponent receptor complex in the retina and superior colliculus (SC) during postnatal development of the rat visual system, and after optic nerve (ON) injury by RT-PCR, immunoblotting and immunofluorescence. Stable mRNA transcription of GDNF and its receptors GFR-alpha1, -alpha2 and Ret was found in retina and SC throughout development into adulthood and after ON transection. Expression of GDNF protein increased during retinal development, declined in adulthood and was further reduced in injured retina. In the SC, GDNF peaked at P0, continuously declined with maturation, and was undetectable in the deafferentiated SC. GFR-alpha1 was abundant in retina and SC throughout, while GFR-alpha2 was not expressed. Since Ret was localized primarily to the vascular compartment, the receptor tyrosine kinase may play a minor role in neuronal GDNF signaling. In summary, we provide evidence for GDNF as survival and guidance factor during development of the retino-tectal projection with differential regulation in early and premature retina and SC. Postlesionally, midbrain targets do not induce GDNF, suggesting that retrograde GDNF is not essential for rescue of adult injured retinal ganglion cells (RGCs).

Oxygen modifies artery differentiation and network morphogenesis in the retinal vasculature.

The mechanisms that control differentiation of immature blood vessels into either arteries or veins are not well understood. Because oxygen tension in arteries is higher than in veins, oxygen has the potential to be an instructive signal for artery/vein (AV) differentiation. We test this hypothesis by exposing newborn mice to moderate hypoxia (10% atmospheric oxygen) and studying AV differentiation in the developing retinal vasculature. Forming retinal arteries fail to express the artery-specific markers Delta-like 4 (Dll4) and EphrinB2 during hypoxia. However, other aspects of AV differentiation are retained such as high levels of alpha smooth muscle actin in arterial mural cells and vein-specific expression of the msr/apj gene. The capillary network between arteries and veins is denser, and capillaries expressing the venous marker msr/apj are found in territories normally occupied by arterial capillaries. Thus, it appears that high oxygen in arterial blood is required for arterial expression of Dll4 and EphrinB2, which could be involved in cell-cell repulsion pathways that dictate the normal segregation of arteries and veins.

Periodic Delta-like 4 expression in developing retinal arteries.

During vascular development, Notch signalling plays important roles in cell-cell communication and cell fate decisions. We studied expression of Notch 1-4 and its ligand Delta-like 4 (Dll4) in the developing retinal vasculature. Dll4 mRNA is strongly expressed in endothelial cells at the very tips of growing vessels ('tip cells') and also in arteries, where it is expressed in a segmented 'tiger's tail' pattern. This implies that developing retinal arteries contain different types of endothelial cells, Dll4-positive and Dll4-negative. The Dll4-positive stripes do not correspond to any obvious morphological property of the vascular network but correlate to some extent with the distribution of platelet derived growth factor B (PDGF-B) mRNA. However, PDGF-B expression is neither as artery-specific nor as clearly segmented as Dll4. Possible target cells for Dll4 signalling are retinal astrocytes (Notch1 positive), arterial pericytes (Notch3 positive) or arterial endothelial cells themselves (Notch4 positive). However, there is no clear reciprocity of Notch and Dll4 expression that allows identification of the interacting cells. Nevertheless, Dll4 stripes are a novel property of immature arteries, the origin and function of which remain to be explained.

PEDF derived from glial Müller cells: a possible regulator of retinal angiogenesis.

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Müller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Müller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Müller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.

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.

VEGF expression by ganglion cells in central retina before formation of the foveal depression in monkey retina: evidence of developmental hypoxia.

In macaque monkeys the foveal depression forms between fetal day (Fd) 105 and birth (Fd 172 of gestation). Before this, the incipient fovea is identified by a photoreceptor layer comprising cones almost exclusively, a multilayered ganglion cell layer (GCL), and a "domed" profile. Vessels are absent from the central retina until late in development, leading to the suggestion that the GCL in the incipient fovea may be transitorily hypoxic. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the hypoxia-inducible transcription factor (HIF)-1, is the principal factor involved in blood vessel growth in the retina. We examined VEGF expression in macaque retinas between Fd 85 and 4 months postnatal. Digoxygenin-labeled riboprobes were generated from a partial-length human cDNA polymerase chain reaction fragment, detected using fluorescence confocal microscopy, and quantified using Scion Image. High levels of VEGF mRNA were detected in astrocytes associated with developing vessels. We also detected strong expression of VEGF mRNA in the GCL at the incipient fovea prior to Fd 105, with peak labeling in the incipient fovea that declined with distance in nasal and temporal directions. By Fd 152 peak labeling was in two bands associated with development of the inner nuclear layer (INL) capillary plexus: in the inner INL where Müller and amacrine cell somas are located, and in the outer INL where horizontal cells are found. The findings suggest that at the incipient fovea the GCL is hypoxic, supporting the hypothesis that the adaptive significance of the fovea centralis is in ensuring adequate oxygen supply to neuronal elements initially located within the avascular region.

Blood supply to the retina and the lens in the gerbil (Meriones unguiculatus).

The blood supply to the retina and the lens in 32 gerbils (Meriones unguiculatus) of both sexes from infancy to maturity was studied under light and stereoscopic microscopes, and a scanning electron microscope. Mercox (CL-2R; Dai Nippon Ink, Tokyo, Japan) was injected into the left ventricle of 30 animals in order to visualize the blood supply to the retina and the lens from the ophthalmic artery. The central retinal artery arises from the ophthalmic artery, passes through the papilla of the optic nerve together with the central retinal vein and penetrates the vitreous space (cavity of the eye) between the lens and the internal limiting membrane of the retina, where it divides into the central branches covering the lens and the parietal branches to supply the retina. The former passes through the hyaloid space after branching several arterioles and then covers the lens like a network from its medial and marginal sides. Different from small experimental animals, the parietal branches, just after separating from the central one, divides into the nasal, dorsal and temporal branches in the vitreous space, each of which then subdivides to distribute across the retina on the inner limiting membrane, then delineates the membrana vasculosa retinae. This basal pattern of vasculization 1 day after birth continues to death. Both the central and parietal branches of the central retinal artery correspond to the branches of the hyaloid artery in embryo and the latter is preserved in adult gerbils.

The development and mature organisation of the end-artery retinal vasculature in a marsupial, the dunnart Sminthopsis crassicaudata.

The end-artery retinal vasculature of a marsupial, the fat-tailed dunnart, was defined by India ink injection and studied in wholemounts. In the adult, the vitreal vasculature supplying the ganglion-cell layer has major paired-vessels in a horizontal H shape. These vessels skirt the area centralis and visual streak that are supplied by fine end-loops. A second vascular layer of uniformly distributed endloops arises from the superficial vessels and lies at the inner nuclear/outer plexiform border. During development, vessels enter the eye via the optic nerve head to form the upper vasculature, assuming an essentially mature arrangement prior to the formation of the area centralis and visual streak. Vessels then descend to form the lower bed. Unlike the cat, the dunnart has retinal vessels that are patent throughout development, their growth is interstitial and reductive remodelling is not seen. A retinal end-artery system may have evolved in marsupials because their precocity requires a vasculature that is functional from early stages of development.

Astrocyte invasion and vasculogenesis in the developing ferret retina.

We studied the time course of astrocyte invasion and blood vessel formation in the developing ferret retina using glial fibrillary acidic protein (GFAP)-immunohistochemistry for astrocytes and isolectin B4 histochemistry for blood vessels. As in other mammals, strongly GFAP positive astrocytes invade the ferret retina from the optic nerve. At birth, strongly GFAP positive astrocytes have reached about 22% of the distance between optic disc and outer retinal edge whereas weakly GFAP positive processes already extend to the edge of the retina. At postnatal days P30-P37 about 82% of the distance between optic disc and outer retinal edge and in the adult 88% of this distance is covered with strongly labelled astrocytes. Superficial blood vessels form from the optic disc. They reach up to about 24% of the retinal radius at birth and grow radially across the retina during further development. At P30-P37, the whole retina is covered with superficial blood vessels. The deep vascular layer forms later (around P30) through sprouting from superficial vessels. The radial pattern of astrocyte and vessel growth from the optic disc is not affected by the formation of the area centralis and visual streak.

Simulation of the growth pattern of the central retinal artery using a fractal modeling technique.

Assisted by computer, we simulated the typical growth pattern of the central retinal artery by a fractal modeling technique. When processing was started with two fertile sides, an iterative processing function generated a ramiform fractal. The branching angle was chosen at random within a certain range. In addition, a feedback restriction forbade any intersection of branches. We demonstrated that the curved shape of the temporal vessels is caused by simultaneous growth of the vessels and the retina. When processing was begun with only one fertile side, the typical vascular pattern of a retinal coloboma was designed.

Central retinal arteries in the full-term newborn: decrease in width and tortuosity during uneventful adaptation.

The fundi of 87 full-term newborns were repeatedly photographed from 2 to 144 h of life. At 2 h of life the width of the temporal arteries in the peripapillary area was about 100 micrometers and that of the nasal arteries about 70 micrometers, and all gradually decreased by about 30% during uneventful adaptation in room air. This decrease did not correlate with concomitant tcpO2 and blood pressure measurements. In most babies the arteries were slightly tortuous at 2 h of life and gradually straightened during adaptation. In some babies, however, there was marked tortuosity at 2 h of life, and this finding was significantly correlated with fetal risk factors. We conclude that marked tortuosity is a sign of passed acute fetal distress and that funduscopy in risk babies should be promoted.