Chapter 18 Retinal and Choroidal Vasculature
Retinal Oxygenation
Introduction
Oxygen is necessary for the existence of mammals because it is required to generate adenosine triphosphate (ATP) oxidatively. Although the partial pressure of oxygen (po2) is 149 mmHg (21% of atmospheric oxygen) at sea level, arterial oxygen content is as low as 75–100 mmHg (10–14%) and tissue po2 is much lower. The oxygen level in inner segments of photoreceptors (mitochondria-rich) after dark adaptation is between 0 and 5 mmHg (0.7%) but up to 20 mmHg in the light. Inner retinal oxygen is normally 20 mmHg, so normoxia depends on the area of retina and dark/light state.1,2 Hypoxia is an oxygen level below normoxia while hyperoxia is achieved by inhaling high levels of oxygen as in the isolette of the neonatal intensive care unit.
The retina is one of the most metabolically active tissues in the body. It has two unique zones of oxygenation.1 The inner retina is supplied with oxygen by the retinal vasculature. The retinal vasculature is autoregulated because it is responsive to changes in systemic oxygen levels, keeping the inner retina at a relatively constant level. If the retinal vasculature is compromised, as in ischemic retinopathies, the retina becomes hypoxic in that area. The outer retina is supplied solely by the choroidal vasculature. Unlike retina, choroidal vessels are not autoregulated, so systemic levels of oxygen control the level of oxygen in choroid. Supply of oxygen to choroid is diminished by stenosis of the ophthalmic artery, which is the branch off the internal carotid that is most likely to be stenosed because it is a right-angle branch.
Comparison of retinal and choroidal vasculatures
Although less than 300 µm apart in distance, the retinal and choroidal vasculatures are vastly different in many attributes in addition to autoregulation. The initial retinal vasculature in human starts forming around 14 weeks’ gestation (WG) by vasculogenesis, development by differentiation and assembly of vascular precursors, angioblasts.3–5 The deep capillary network forms after 20 WG by angiogenesis, development by migration, and proliferation of endothelial cells from existing blood vessels. The driving force for vascular development is physiological hypoxia; metabolic requirements of developing neurons are only met by stimulating the development of a retinal vasculature.6,7 It is mostly a bilayered system, a superficial network, and a deep capillary plexus; however, there are multiple layers of capillaries in the peripapillary region, the radial peripapillary capillaries. There is only one layer of blood vessels in the periphery at the ora serrata where the retina thins to 100 µm. The retinal vasculature is supplied with blood directly by the central retinal artery in humans. The retinal vasculature has a traditional end-arterial hierarchy: arteries branch to arterioles, which supply a capillary network that is drained by venules, and then veins remove the blood from the retina (Fig. 18.1). The retinal vasculature forms the inner blood–retinal barrier (BRB), restricting passage of molecules that do not have receptors or transporters on the luminal surface of the endothelial cells. Capillaries have a lumen diameter of 3.5–6 µm, permitting passage of red blood cells only after deformation of their disc shape. The retinal capillaries and venules have perivascular pericytes and the retina has the highest endothelial cell-to-pericyte ratio in the body, 1 : 1.8
The choroidal vasculature forms well before the retinal vessels (6–9 WG), although its maturation is only completed after 20 WG. It develops by hemovasculogenesis, formation of blood cells and blood vessel cells from a common progenitor, the hemangioblast.9 The choroidal vasculature provides oxygen and nutrients to the photoreceptors. The capillary system, the choriocapillaris, lies directly under the Bruch’s membrane, while intermediate and large blood vessels of the system lie posterior to the capillaries. The short and long ciliary arteries supply blood to the choroidal vasculature while 4–6 vortex veins remove blood from this vast system. Unlike the retina, the hierarchy in choroid is lobular, similar to kidney glomeruli (Fig. 18.1). The lobules change in shape, vascular density, and size depending upon area and the location of feeding arterioles and draining venules also varies by geographic location of the lobule.10 The capillaries are broad and flat, having luminal diameters ranging from 10 to 38 µm in diameter. Another major difference from retina is that the capillaries are fenestrated, allowing the passage of small molecules and solutes through these 60–70-nm pores. The choriocapillaris is sided in that the majority of the fenestrations are on the retinal side as well as all three types of vascular endothelial growth factor (VEGF) receptors.11 Pericytes, however, are mostly on the scleral side of these capillaries. Control of vascular tone in choroid may be accomplished by mast cells, which lie abluminal to arteries and arterioles, or choroidal ganglion cells.12
History of retinal ischemia
Ischemia is the restriction in oxygen supply without considering actual levels of oxygen. Michaelson13,14 and Wise15 hypothesized that areas of vascular loss in retina must be hypoxic because the high metabolic rate requires a continuous supply of oxygen. They observed that neovascularization always formed adjacent to these nonperfused areas and, therefore, an angiogenic factor must be produced by the hypoxic retina. They hypothesized that this factor X must be hypoxia-inducible and diffusible. Subsequently, oxygen was measured directly in retinas of several species and it demonstrated that nonperfused areas were indeed hypoxic.1,2 It was not until 1989 that factor X was discovered, purified, and characterized as vascular endothelial growth factor (VEGF).16 This factor was first shown to be responsible for the increased vascular permeability seen in some retinopathies.17
Normoxia
The studies of Wangsa-Wirawan and Linsenmeier1 and Yu and Cringle2 using oxygen electrodes directly assessed oxygen levels from choroid to vitreous in various species. The oxygen tension is around 70 mmHg in choroid and plummets to zero at the inner segments in the dark (Fig. 18.2). Inner retina is around 10–20 mmHg. There are regional variations in oxygen concentration within the retina. Yu et al.18 showed that oxygen consumption in outer retina is highest in the parafoveal region while inner retinal oxygen in the fovea (approximately 5 mmHg) reflected the lack of a retinal vasculature and the predominantly choroidal source of oxygen.
Hyperoxia
Life in utero is hypoxic, so when a child is born prematurely, the normoxic environment is actually hyperoxic. Prematurely born children are placed in 40% oxygen, making their tissue further hyperoxic, which yields vaso-obliteration (endothelial cells die and pericytes and progenitors survive).19 The only direct measurements of oxygen in a model of retinopathy of prematurity (ROP) were performed by Ernest and Goldstick.20 They found in kitten after 80–90% O2 that preretinal po2 over avascular retina was close to zero but was normal over vascularized retina. Vaso-obliteration from hyperoxia does not occur in the choroid of humans and dogs19 but does occur when rats are exposed to hyperoxia.21 Loss of vasculature in vaso-obliteration makes the retina hypoxic when the child is returned to room air. Exposure of the adult vasculature to hyperoxia causes constriction but not vaso-obliteration. During hyperoxia breathing (100% oxygen), the inner retinal po2 remains unchanged due to autoregulation while the choroidal po2 rises to 250 mmHg in cat22 and 220 mmHg in the minipig, due to a lack of metabolic control of the choroidal vasculature.23
Hypoxia
Complex homeostatic mechanisms are designed to maintain O2 concentration in each cell within a narrow range. While O2 consumption increases with the metabolic activity of the organism, exposure to O2 must be limited due to the potentially damaging effects of reactive oxygen species (ROS). Hypoxia, the state of low oxygen concentration, promotes the formation of blood vessels and is important for the formation of a vascular system in embryos.24 Disease occurs when the retina and choroid are deprived of adequate oxygen supply; this can also be described as a mismatch of oxygen supply versus demand at the cellular level within ocular tissues.
The blood O2-carrying capacity is maintained by the O2-regulated production of erythropoietin (EPO), which stimulates the proliferation and survival of red blood cell progenitors. Semenza and coworkers25,26 performed seminal studies to identify hypoxia-inducible factor-1 (HIF-1). HIF-1 orchestrates a pleiotropic adaptive response to hypoxia by inducing the expression of more than 100 genes encoding glycolytic enzymes and glucose transporters (thereby facilitating the glycolytic switch in energy metabolism typically observed under hypoxic conditions), matrix metalloproteinases, and angiogenic, mitogenic, and survival factors, including EPO.27,28 Other molecules upregulated by HIF-1 that have profound effects on vasculature include 5’ nucleotidase, an enzyme that is the major source of the potent vasodilator adenosine in the body, and VEGF. HIFs are vital to development and, in mammals, deletion of the HIF-1 genes results in perinatal death. HIF-1 is expressed in all cell types and functions as a master regulator of oxygen homeostasis by playing critical roles in embryonic development and postnatal physiology.
Hypoxia-inducible factor
The activity of HIF depends on the intracellular levels of its inducible alpha subunit. In the presence of oxygen, HIF-1α is hydroxylated on two critical proline residues (Pro402 and Pro564) in the so-called oxygen-dependent degradation domain. Three prolyl hydroxylases have been identified in mammalian cells and use O2 as a substrate to generate 4-hydroxyproline at residues 402 and/or 564 of HIF-1α. The hydroxylation reaction also requires 2-oxoglutarate (α-ketoglutarate) as a substrate and generates succinate as a side product. These prolyl hydroxylases have a high Km for O2 that is slightly above atmospheric concentration; thus O2 is rate-limiting for enzymatic activity under physiological conditions and any change in cellular O2 concentration is directly transduced into changes in the rate of HIF-1α hydroxylation.29
Factor-inhibiting HIF-1 (FIH-1), which was identified in a yeast two-hybrid screen as a protein that interacts with, and inhibits the activity of the HIF-1α transactivation domain,30 functions as asparaginyl hydroxylase.31 As in the case of the prolyl hydroxylases, FIH-1 appears to use O2 and 2-oxoglutarate,32 although it has a Km for O2 that is three times lower than the prolyl hydroxylases.33 Hydroxylation provides a mechanism for regulating protein–protein interactions, similar to the effect of phosphorylation and other posttranslational modifications. However, this hydroxylation occurs in an O2-dependent manner, thus establishing a direct link between cellular oxygenation and HIF-1 activity. Following HIF-1α hydroxylation, the protein becomes targeted for ubiquitination by an E3 ligase complex (including the von Hippel–Lindau (VHL) tumor suppressor protein) and subsequent proteasomal degradation.
Under hypoxic conditions, the HIF prolyl-hydroxylases are inhibited, because these HIF prolyl-hydroxylases utilize oxygen as a cosubstrate. Hypoxia results in an increase in succinate, due to inhibition of the electron transport chain in the mitochondria, which serves to inhibit further HIF prolyl-hydroxylase activity. When stabilized by hypoxic conditions, HIF increases the expression of critical genes that promote survival in low-oxygen conditions, including glycolytic enzymes, which allow ATP synthesis in an oxygen-independent manner. HIF activates the transcription of genes encoding secreted signaling molecules, including angiogenic growth factors and survival factors, cell surface receptors, extracellular matrix proteins and modifying enzymes, transcription factors, cytoskeletal proteins, proapoptotic proteins, and glucose transporters and glycolytic enzymes (Fig. 18.3).29
HIF-induced VEGF, stromal-derived factor-1 (SDF-1), and EPO promote neovascularization. HIF-1 acts by binding to HIF-responsive elements in promoters that contain the sequence NCGTG, which is present in the promoters for VEGF, SDF-1, EPO, and many other genes. In addition to hypoxia, other factors such as nuclear factor κB (NF-κB) modulate HIF-1α expression in the presence of normal oxygen pressure. Thus, conditions such as tissue inflammation can lead to local HIF-1α expression.34 HIF-1 DNA-binding activity and target gene expression are induced in cells exposed not only to hypoxia but also to the iron chelator desferrioxamine or to cobalt chloride.35
A structurally and functionally related protein to HIF-1α, designated HIF-2α, is the product of the EPAS1 gene. HIF-2α can also heterodimerize with HIF-1ß.36 HIF-1α:HIF-1ß and HIF-2α:HIF-1ß heterodimers have overlapping yet distinct target gene specificities.37 HIF-2α, unlike HIF-1α, is not expressed in all cell types and HIF-2α can be inactivated by cytoplasmic sequestration. This “compartmentalization” of oxygen-sensitive signaling components also influences the hypoxic response.38,39
HIF deficiency and its resultant pathology
O2 delivery to cells of the developing embryo becomes limited by diffusion such that establishment of a functioning circulatory system is required for embryonic survival by embryonic day 9 (E9) in the mouse. In wild-type mouse embryos, HIF-1α expression increases dramatically between E8.5 and E9.5, whereas embryos that lack HIF-1α expression die between E9.5 and E10.5 and show cardiac malformations, vascular regression, and massive cell death.40 Complete HIF-2α deficiency is also associated with embryonic lethality41 and because the embryos survive longer than HIF-1a–/– mice, effects on multiple organ systems can be demonstrated.42
Complete HIF-1α deficiency results in developmental defects; however, partial HIF-1α deficiency is sufficient to result in impaired responses to physiological stimuli. A particularly dramatic example is the loss of O2 sensing in the carotid body of HIF-1a+/– mice.43 Although the carotid bodies are anatomically and histologically normal and depolarize normally in response to cyanide application, they show essentially no response to hypoxia. Thus partial HIF-1α deficiency in the carotid body results in a complete loss of the ability to sense and/or respond to changes in the arterial Po2 by stimulation of the central nervous system cardiorespiratory centers. The HIF-1 target genes that are critical for O2 sensing and/or efferent responses by the carotid body have not been identified.
Mice with HIF-1α conditionally knocked out using PAX6-Cre have delayed development of the outer retinal plexus but not the superficial or deep plexus.44 However, when HIF-1α was knocked down only in Müller cells using a Cre-LOX system, and the animals were made diabetic with streptozotocin, vascular permeability in retina was reduced and leukostasis and overproduction of VEGF and intercellular adhesion molecule (ICAM)-1 were attenuated in adult mice.45
Another dramatic phenotype is the complete inability of HIF-1α–/– myeloid cells (granulocytes and macrophages) to respond to inflammatory stimuli.46 Myeloid cells are dependent on glycolysis for ATP generation, perhaps reflecting the hypoxic microenvironment that is often associated with inflammation and infection. HIF-1α deficiency results in ATP deficiency, which impairs critical myeloid cell functions such as aggregation, motility, invasion, and bacterial killing. HIF-1 also plays critical roles in B-lymphocyte development47 and T-lymphocyte activation.48
HIF-activated genes relevant to physiological and pathological ocular angiogenesis
The paragraphs above provide a brief summary of the critical role of HIF-1α in oxygen sensing, development, and physiology. HIF-1α plays an equally important role in disease pathophysiology, including retinal diseases. As a result, there is considerable interest in HIF-1 α as a therapeutic target.49 In cardiovascular diseases, increased HIF-1α activity induced as a result of HIF-1α gene therapy,50 small-molecule inhibitors of prolyl hydroxylase activity,51 or inhibitors of HIF-1α–VHL interaction52 may provide a means to stimulate neovascularization in ischemic tissue. In contrast, small-molecule inhibitors of HIF-1 activity may be useful as antiangiogenic agents. However, because HIF-1α functions as a global regulator of oxygen homeostasis, it may not be a useful therapeutic target if the treatment results in unintended and undesirable side-effects.
An alternative therapeutic approach that may be particularly relevant to the treatment of ocular pathology is to focus on modulation of HIF-1α target genes. However, the protein products of these target genes must also be delivered in a precise and perfectly timed manner. EPO is an oxygen-regulated hormone stimulating erythrocyte production and is critical for retinal angiogenesis. Increasing EPO expression in phase 1 of the murine ROP model (postnatal days 7–12) is protective and results in less neovascularization during phase 2 (postnatal days 12–17).53 In contrast, EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy (phase 2) and inhibition of retinal EPO mRNA expression with RNA interference results in suppressed retinal neovascularization.53
The best-known gene activated by HIF-1α is VEGF, first identified as a potent promoter of vascular permeability17 and endothelial cell proliferation.15 VEGF has become known as a master regulator of angiogenesis.54 Tight control of physiologic VEGF levels is required for proper embryological development.55 Although it was initially thought that the postembryonic role of VEGF was restricted to a few processes, it is now quite clear that VEGF acts as a pluripotent growth factor essential for a wide variety of physiological processes,56 including maintenance of the adult microvasculature,57 neuronal survival,58 and trophic maintenance of ocular tissues.
VEGF in health and in ocular disease
VEGF is produced by many cell types in the retina, including retinal pigment epithelium (RPE),59 vascular endothelial cells,60 pericytes,60 retinal neurons,61 Müller cells,61 and astrocytes,62 suggesting that VEGF has important functions in ocular homeostasis. RPE-secreted VEGF plays an important role in maintaining the choriocapillaris.11,63,64 VEGF secretion by retinal cells and the RPE is stimulated in response to hypoxia.60 VEGF administration protects retinal neurons from apoptosis.65 Moreover, chronic VEGF inhibition can lead to a significant loss of retinal ganglion cells in normal adult animals.65
While VEGF is critical to maintaining normal ocular function, overproduction of VEGF is deleterious. Elevated levels of VEGF have been strongly implicated in the pathogenesis of ocular neovascular diseases such as neovascular age-related macular degeneration (NV in AMD)66 and proliferative diabetic retinopathy67 as well as diabetic macular edema.68 Elevated VEGF levels are observed in central and branch retinal vein occlusion (CRVO and BRVO),69 neovascular glaucoma,70 and ROP.71 Blocking VEGF action is now an established strategy for the treatment of NV in AMD, with two agents (the RNA aptamer pegaptanib sodium72 and the humanized murine monoclonal antibody antigen-binding fragment ranibizumab73) having received regulatory approval for the intravitreal treatment of NV in AMD.
While current standard of care for NV in AMD uses intravitreal delivery of an anti-VEGF agent on a repeated basis, this routine clinical practice does not address all the nuances of VEGF biology. The biology of the VEGF proteins is extremely complex. The VEGF family members are part of a superfamily of cysteine knot proteins and include VEGF-B, -C, -D, and placental growth factor (PlGF). Alternative splicing events for VEGFA give rise to at least 14 subtypes of VEGF, namely, VEGF111, VEGF121, VEGF121b, VEGF145, VEGF145b, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF183b, VEGF189, VEGF189b,74 and VEGF206.75,76 Following the discovery of the antiangiogenic isoform of VEGF, VEGF165b, and its associated family of isoforms, a further layer of complexity was added to understanding the regulation of VEGF.
All VEGF isoforms are essential regulators of angiogenesis and vascular permeability. VEGFs elicit their intracellular activities via the activation of two receptor tyrosine kinases (RTKs): VEGFR-1 and -2. VEGFR-1, a high-affinity fms-like tyrosine kinase-1,77 and VEGFR-2, a kinase insert domain-containing receptor,78 are transmembrane glycoproteins consisting of a seven-tandem immunoglobulin-like domains, which serves as the extracellular ligand-binding region, a single-transmembrane domain, and a cytoplasmic domain consisting of two tyrosine kinase catalytic domains. Moreover, it has also been reported that a family of cell surface glycoproteins, particularly neuropilin-1, act as isoform-specific coreceptors for VEGF-A.79 The VEGF family and their respective receptors are outlined in Fig. 18.4.
Ligand binding to the extracellular domain of VEGFR-2 results in a maximal increase of kinase activity following the induction of receptor dimerization and subsequent phosphorylation of tyrosine residues on the intracellular domain of the receptor.80 This event is crucial for the recruitment of additional signaling molecules that contain Src homology 2 or phosphotyrosine binding domains, which mediate further downstream signaling cascades.81 The association of RTKs with coreceptors, such as NP-1, in the case of VEGFR-2:VEGF165 signaling/interaction, can enhance the functional signal transduction and facilitate diverse cellular responses.80 VEGFR1/R2 signaling activates RAS, raf1, MEK1, and ERK1/ERK2 and stimulates PI3K/AKT/PKCz/MAPK pathways to mediate proliferation, migration, and cell survival (Fig. 18.4).
VEGFR-2 is the major mediator of angiogenic signaling in endothelial cells and is required for de novo vessel formation, vasculogenesis, and for angiogenesis, the formation of vessels from pre-existing vasculature.82 The pathways leading to VEGFR internalization and the role of receptor degradation in VEGF signaling remain controversial and differ for VEGFR-1 and -2. VEGFRs generate signal output at the plasma membrane and on their way to degradation through endocytic vesicles,83 whereas, in unstimulated cells, VEGFR-2 is predominantly located in recycling endosomes identified by Rab4 and/or Rab5.84 VEGFR internalization is clathrin-mediated and transport is further directed by the endosomal sorting complex required for transport proteins.85 VEGFR signaling is also regulated by ubiquitination, not only of the receptor itself, but also of receptor-associated signaling molecules.86 Specific VEGFR trafficking regulates biological output, as shown for arterial morphogenesis, for example.87
The molecular basis for ligand specificity of VEGFR signaling is poorly understood. It is well accepted however that VEGF receptors can associate with distinct coreceptors such as neuropilins, integrins, semaphorins, or heparan sulfate glycosaminoglycans, and engage distinct signaling molecules giving rise to specific signal output. Ligand-specific signaling may also result from receptor trafficking to specific cellular compartments, including the nucleus,88 where receptors encounter distinct signaling molecules.89
In addition to EPO and VEGF, SDF-1 is hypoxia-regulated and ischemic tissues express high levels of SDF-1 to recruit reparative cells to the injured region. SDF-1 activation of either CXCR-4 or CXCR-7 results in stimulation of the Ras/Raf/Mek/ERK pathway and the PI3K/Akt pathway to promote endothelial cell proliferation and neovascularization. Thus ligand–receptor interaction of VEGF to VEGF-R1 and VEGF-R2 and SDF-1 to CXCR4/CXCR-7 and their subsequent internalization sets in motion the cascade of cellular effects of VEGF and SDF-1 (Fig. 18.4). The VEGF and SDF-1 signaling pathways appear to be intimately connected with HIF-1 activation (Fig. 18.3), as the promoters of each of these factors contain a HIF response element. Because hypoxic tissue releases SDF-1 and VEGF, varying O2 concentrations would have an effect on expression of the receptors for these factors.
Bone marrow-derived progenitor cells (BMPC) and vascular repair
In conditions like diabetic retinopathy and ROP, areas of retinal vasodegeneration occur and lead to retinal ischemia which in turn induces the expression of hypoxia-regulated angiogenic factors. Typically, BMPCs robustly respond to these factors, including VEGF and SDF-1.90
Importantly, all hypoxia-regulated angiogenic factors are modulators of bone marrow-derived stem cells. Specifically, hematopoietic stem cells and other BMPCs reside in the bone marrow and are mobilized into the peripheral circulation by increased levels of EPO, SDF-1, and VEGF released by the ischemic tissue. Hematopoietic stem cell/BMPC mobilization occurs in response to vascular injury throughout the body and, when functioning properly, leads to revascularization of injured areas.91 In healthy individuals, bone marrow-derived CD34+ endothelial progenitor cells as well as other BMPC successfully orchestrate the reparative process. These BMPC demonstrate a limited ability to differentiate directly into endothelial cells and form components of new blood vessels by vasculogenesis, but show marked ability to provide paracrine support for the resident vasculature. This paracrine support facilitates resident endothelial cell recovery.
Disease-associated BMPC dysfunction
In diabetes, for example, BMPC are dramatically altered and cannot facilitate the repair process. Diabetic individuals have fewer circulating CD34+ cells and an increased number of inflammatory BMPC such as CD14+ cells than nondiabetics.92–94 This diabetes-related bone marrow dysfunction is closely linked to the impaired healing response experienced by many diabetic patients and to the vasodegenerative aspect of diabetic macro- and microvascular complications.94–96 Diabetes-induced BMPC defects occur in part due to uncoupling of nitric oxide synthases, enhanced NADPH oxidase activity, and increased generation of ROS such as superoxide and peroxynitrite (ONOO-)97 within BMPC. While stem and progenitor cells are deemed more resistant to oxidative stress,98 the highly oxidative diabetic milieu has a clearly detrimental effect on the function of these cells.99 Prolonged oxidant exposure reduces reparative function100 by impairing antioxidant defense enzymes. Previously, we and others have shown that diabetic CD34+ cells exhibit decreased migration and adhesion activities in vitro, and consequently reduce recruitment to areas of injury.96 In addition to oxidative stress, other key mechanisms implicated in diabetes-induced BMPC dysfunction include a reduction of cathepsin L activity101 and an upregulation of thrombospondin-1.100,102 While these functional defects are profound, strategies that successfully reverse BMPC defects in diabetics have included: (1) enhancement of angiogenic stimulus by increasing BMPC mobilization using granulocyte colony-stimulating factor and targeting SDF-1103; (2) use of nitric oxide donors to correct migration and promote cell deformability104; (3) enhancing cell interactions with substrate proteins to increase attachment to basement membranes105
