Tailoi Chan-Ling

Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia

We have studied the role of the hypoxia-inducible angiogenic growth factor vascular endothelial growth factor (VEGF) in the induction and control of vessel growth in the developing retina of rats and cats, using in situ hybridization techniques. VEGF is expressed successively in two layers of neural retina, the innermost (axon) layer and the inner nuclear layer (INL). In the axon layer, VEGF is expressed transiently by astrocytes as they spread across the layer, closely preceding the formation of superficial vessels. In the INL, VEGF is expressed transiently by somas at the middle of the layer (presumably Muller cells), closely preceding the formation of the deep layer of retinal vessels. We propose that hypoxia caused by the onset of neuronal activity is detected by strategically located populations of neuroglia, first astrocytes, then Muller cells. In response they secrete VEGF, inducing formation of the superficial and deep layers of retinal vessels, respectively. As the vessels become patent, they relieve the hypoxic stimulus, so vessel formation is matched to oxygen demand. This hypothesis was tested experimentally in three ways. Expression of the high affinity flk-1 receptor for VEGF was demonstrated in newly formed retinal vessels, confirming that the secreted VEGF acts on the vessels, in a paracrine fashion. Direct hypoxic regulation of VEGF expression by macroglia was demonstrated in primary cultures of astrocytes and in cells of a glioma line. Hypoxic regulation of VEGF expression in the intact developing retina was demonstrated by showing that oxygen-enriched atmospheres that inhibit vessel formation also suppress endogenous VEGF production.

Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease.

We review the cellular and physiological mechanisms responsible for the regulation of blood flow in the retina and choroid in health and disease. Due to the intrinsic light sensitivity of the retina and the direct visual accessibility of fundus blood vessels, the eye offers unique opportunities for the non-invasive investigation of mechanisms of blood flow regulation. The ability of the retinal vasculature to regulate its blood flow is contrasted with the far more restricted ability of the choroidal circulation to regulate its blood flow by virtue of the absence of glial cells, the markedly reduced pericyte ensheathment of the choroidal vasculature, and the lack of intermediate filaments in choroidal pericytes. We review the cellular and molecular components of the neurovascular unit in the retina and choroid, techniques for monitoring retinal and choroidal blood flow, responses of the retinal and choroidal circulation to light stimulation, the role of capillaries, astrocytes and pericytes in regulating blood flow, putative signaling mechanisms mediating neurovascular coupling in the retina, and changes that occur in the retinal and choroidal circulation during diabetic retinopathy, age-related macular degeneration, glaucoma, and Alzheimers disease. We close by discussing issues that remain to be explored.

The role of müller cells in the formation of the blood-retinal barrier

We have compared the ability of Müller cells and astrocytes to induce the formation of barrier properties in blood vessels. Müller cells cultured from the rabbit retina, and astrocytes and meningeal cells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Müller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Müller cells play a major role in the formation of barrier properties in retinal vessels.

Development of retinal vasculature in the cat: processes and mechanisms.

Two principal processes can be distinguished in the development of the retinal circulation in the cat. One process, which forms most of the inner layer of vasculature, involves three stages. First, beginning prior to E (embryonic day) 26, spindle cells of mesenchymal origin spread over the inner surface of the retina. Second, beginning at approximately E48, a network of coarse capillaries forms, apparently derived from spindle cells. Third, major vessels differentiate from the capillary plexus, and the capillaries become thinner and more widely spaced. All three stages begin at the optic disc and spread towards the margin of the retina. The other process involves budding of capillary sized vessels from existing vasculature. This process forms the inner layer of vasculature at the area centralis, the outer layer of vasculature, and the radial peripapillary capillaries. It begins between P (postnatal day) 7 and P10 at the area centralis and spreads to the margins of the retina. The radial peripapillary capillaries form at a later stage (P20). The different topographies of the two processes suggest that they are controlled by distinct mechanisms. In the first process, the formation of vessels follows a pattern set by the early migration of spindle cells. In the second process, the vessels form in a pattern determined by the metabolic needs of the developing retina.

IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit+), stem cell antigen-1 (sca-1+), hematopoietic stem cell (HSC), or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions.

Glial, vascular, and neuronal cytogenesis in whole‐mounted cat retina

A method was developed for detecting cytogenesis in retinal whole‐mount preparations by bromodeoxyuridine (BrdU) immunohistochemistry. Because BrdU is a nonspecific marker that labels all cells in the S phase of the cell cycle, it is ideally combined with other cell‐specific markers to study the cytogenesis of specific cell types. Double‐label protocols to visualize mitotically active astrocytes and cells associated with the forming vasculature have been developed and applied to the retina. This approach revealed that, during normal development of the kitten retina, vascular mitogenesis occurs predominantly in the ganglion cell and nerve fiber layers, where the inner retinal plexus is formed by a process involving transformation of mesenchymal precursor cells and division of vascular endothelial cells. The peak density of vascular mitogenesis moved in a central‐to‐peripheral manner and was associated with the leading edge of the forming capillary plexus. A small number of dividing vascular endothelial cells was also associated with angiogenesis, the process responsible for the formation of the outer retinal plexus, vessels at the area centralis, and the radial peripapillary capillaries. Cytogenesis associated with astrocytes occurred in the ganglion cell and nerve fiber layers but was apparent predominantly at or close to the optic nerve head. Confirming earlier studies, neuronal mitogenesis was shown to occur predominantly at the ventricular zone, first at the area centralis and spreading peripherally with increasing maturity. A second region of neuronal cytogenesis, at the subventricular zone, was also apparent. Tissue hyperoxia decreased the rate of vasculogenic cell division but had no apparent effect on neurogenic or astrocytic cell division. Four distinct zones of cell generation were therefore identified within the retina, each associated with either glial, vascular, or neuronal cytogenesis. Thus, BrdU immunohistochemistry in whole‐mounted retinal preparations offers a fast and reliable alternative to [3H]thymidine autoradiography for the study of the topography of cytogenesis during development. Microsc Res Tech 36:1–l6, 1997.

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple‐label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP‐ APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin‐/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2‐/vimentin‐/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.

Early activation of microglia in the pathogenesis of fatal murine cerebral malaria

Microglia are pluripotent members of the macrophage/monocyte lineage that can respond in several ways to pathological changes in the central nervous system. To determine their role in the pathogenesis of fatal murine cerebral malaria (FMCM) we have conducted a detailed study of the changes in morphology and distribution of retinal microglia during the progression of the disease. Adult CBA/T6 mice were inoculated with Plasmodium berghei ANKA. These mice died 7 days post inoculation (p.i.) with the parasite while exhibiting cerebral symptoms, increased permeability of the blood‐brain barrier, and monocyte adherence to the vascular endothelium. Mice were injected i.v. with Monastral blue 2 h prior to sacrifice to identify “activated” monocytes, and their isolated retinae were incubated with the Griffonia simplicifolia (GS) lectin or reacted for the nucleoside diphosphatase enzyme to visualize microglia and the vasculature. Changes in microglial morphology were seen within 2–3 days p.i., that is, at least 3 days prior to the onset of cerebral symptoms and 4 days before death. Morphological changes included retraction of ramified processes, soma enlargement, an increasingly amoeboid appearance, and vacuolation. There was also increased staining intensity and redistribution of “activated” microglia toward retinal vessels, but no increase in density of NDPase‐positive cells. The GS lectin only labeled a small population of microglia in the uninfected adult mouse retina. However, there was a striking increase in the focal density of GS‐positive microglia during the progression of the disease. Extravasation of monocytes also was observed prior to the onset of cerebral symptoms. These results provide the first evidence that microglial activation is a critical component of the pathological process during FMCM.

Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4–2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1−/− with Ndfip1+/+ mice exposed to Fe reveals that Ndfip1−/− brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.