Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

(a)Basal surface

Rb-/-

Figure 4 Uncoupling cell birth and cell-cycle exit. (a) In the WT retina, the RPC nucleus/cell body changes position as the cell progresses through the cell cycle. This process is termed interkinetic nuclear migration (INM). Following cell birth, the apical process begins to retract as the newborn RTC moves to its final destination and undergoes terminal differentiation (TD) Green and red nuclei depict whether the cell is dividing or post mitotic, respectively. Yellow cytoplasm indicates induction of a distinct transcriptome in the RTC. (b) In the absence of Rb, RPC division is unaffected, but differentiating RTCs divide ectopically (note green, instead of red, nucleus). However, the birth transcriptome (yellow cytoplasm) is activated. The response of different RTCs to Rb loss is cell-type specific. Some (as depicted here) survive, terminally differentiate and exit division via Rb-independent means. During ectopic division, these cells are at risk for neoplastic transformation (not shown). Other cell types (e.g., ganglion cells) escape transformation by undergoing apoptosis (not shown).

important to ensure proper migration of forebrain neurons against independent of cell cycle/apoptosis activities. Amacrine cells are one of a subset that survive loss of Rb proteins, and most escape tumorigenesis not by death, but by Rb-independent cell-cycle escape (Figure 4). However, rare Rb/p107-null or Rb/p130-null amacrine cells – likely through post-pocket protein events that override this arrest – can form sporadic retinoblastoma. Human retinoblastoma does not require p107 or p130 loss, likely reflecting broader expression of these proteins in other species. Intriguingly, long-term ectopic division of differentiating cells in fly tissues also requires disruption of multiple cell-cycle regulators. Ectopically dividing murine horizontal cells are also resistant to apoptosis, but they are better protected against transformation and require loss of Rb, p130, and one allele of p107 to form tumors. Natural resistance to apoptosis is an attractive feature for a cancer cell-of-origin, especially one like retinoblastoma that requires fewer rate-limiting events than adult cancers. Several post-Rb events have been identified in human retinoblastoma and likely facilitate progression past a benign retinoma state, which ends in senescence.

In summary, pocket proteins apparently do not regulate the cell cycle in normal RPCs, but are poised to act in differentiating RTCs where Rb is required to quench E2f1 activity (Figures 3 and 4). p107 and p130, although non-

essential, act as backups when Rb is removed. The CKIs p19Ink4d and p27Kip1 may play an important role in acti-

vating Rb proteins in differentiating RTCs (Figure 3). Pocket protein loss creates a dangerous state where ectopically dividing RTCs risk neoplastic transformation. This risk is countered in some cells by apoptosis and others by Rb-independent means of cell-cycle exit.

Ink4 CKIs and p19Arf in Retinal

Development

Cdkn1c encoding p18Ink4c is expressed weakly in the

embryonic NBL, but is dispenable for retinal development. Cdkn2b, which encodes p15Ink4b, lies adjacent to

Cdkn2a, which encodes two transcripts that have distinct first, but shared downstream, exons and encode p16Ink4a and the p53-activating protein p19Arf (p14ARF in humans) from different reading frames. p15Ink4b expression has not been reported in the retina and, while deleting both Cdkn2b/2a loci (which removes all three proteins) renders mice extremely susceptible to tumorigenesis, eye defects in addition to those seen in Cdkn2a or p19Arf-null mice were not described. p19Arf is expressed in embryonic vitreal pericytes where it represses platelet-derived growth factor receptor beta (PDGFRB) expression independent of double minute 2 (MDM2) or p53, limiting expansion of these endothelial support cells, thus its absence triggers abnormal expansion and severe defects

in the adult eye. As expected, Cdkn2a-null mice, lacking both p16Ink4a and p19Arf, also have this defect, but no

obvious retinal defects – consistent with the absence of these proteins in RPCs. The fourth Ink4 protein – p19Ink4d – is encoded by Cdkn2d. Its expression pattern is consistent with a role in facilitating cell-cycle exit (Figure 3) and, indeed, null mice show abnormal division followed by elevated apoptosis. The defects may be a milder version of those seen in the absence of Rb or following overexpression of E2f1 or cyclin D1 in differentiating retinal neurons.

Cip/Kip CKIs in Retinal Development

Some p21Cip – encoded by Cdkn1a – is expressed in the WT retina, which increases in the absence of Rb, implying a context-specific role in retinal cell-cycle control. p21Cip absence alone does not affect retinal development, so it would be interesting to know its effect when combined with Rb loss.

In the embryonic retina, a few cells express p57Kip2 (e.g., 3%, E14.5) and its loss triggers extra division, which may reflect extra RPCs and/or ectopically dividing

RTCs; the latter fits the observation that p57Kip2 is expressed in late G1 or G0. Expression ceases around P0, and is reactivated in a subset of postmitotic amacrine cells, consistent with a role for p57Kip2 in differentiation.

The most influential Cip/Kip CKI in the retina is p27Kip1 (Figure 3), as suggested by its broader expression

pattern in mouse and human retina (e.g., 50%, E14.5). p27Kip1 mRNA is high in mouse RPCs and low in differ-

entiating RTCs but vice versa for protein, implying that rapid translation and mRNA degradation parallels differentiation. In human RTCs, p27Kip1 expression seems, in general, to precede Rb protein expression, suggesting that

a dual wave of inhibitors is employed to shut division down. p27Kip1 is detected at G2 in RPCs, earlier than p57Kip2, consistent with an early role in promoting cell-

cycle exit in newborn RTCs. The Cdkn1b-null retina has excess dividing cells at least until P10, which could reflect extra RPCs and/or ectopically dividing differentiating RTCs.

Some adult p27Kip1-deficient retinas contain focal hyperplastic lesions, possibly due to reactive gliosis. These lesions are more severe when the Cdkn1b gene is

replaced by an altered protein (p27CK ) that cannot bind cyclin–Cdk complexes. p27+/CK mice do not get retino-

blastoma, but do develop lung tumors. The increased phenotypic severity in p27+/CK mice relative to p27Kip1-null mice reveals that p27Kip1, when freed from cyclin–Cdks,

has a dominant disruptive function. This activity may relate to the cytoplasmic role of p27Kip1 in regulating the Rho-

Rock signaling pathway which, intriguingly, is also targeted by p21Cip and p57Kip2. p27Kip1 has other cytoplasmic activ-

ities such as binding the microtubule regulator stathmin. p21Cip and 27Kip are distributed between the cytoplasm and

nucleus in mouse retina.

Cell-cycle defects in the p19Ink4d- or p27Kip1-deficient retinas are enhanced when both are missing, consistent with cooperative Rb activation and Cdk inactivation to promote cell-cycle exit. Apoptosis and dysplasia are also more severe. As in the Rb / retina, deleting p53 does not rescue apoptosis, yet surprisingly it does reverse the dysplasia.

E2Fs in Retinal Development

Multiple E2f mRNAs have been detected in the retina, and protein expression has been confirmed for E2f1 as well as both a and b isoforms of E2f3. Deleting E2f1 slows RPC division approximately twofold, whereas E2f3 loss has no effect, suggesting redundancy. The superior role for E2f1 in RPCs differs from that in MEFs where E2f3 is more important. As noted above, E2f1 drives ectopic division in differentiating Rb / RTCs and transgenic E2f1 expression in photoreceptors also impairs cell-cycle exit. E2f3a perturbs differentiation in some Rb / amacrine

cells, which is similar to its effect in the Rb / forebrain. E2f4 loss affects Shh expression in the telencephalon, but its role in the retina, similar to other E2fs, awaits further study.

Separating Rate, Birth, and Exit

The above discussion summarizes how cell-cycle regulators affect RPC expansion and RTC cell-cycle exit. We now turn to how the factors that promote cell birth influence the cell-cycle machinery. At this point, we encourage the reader to review the glossary definition of the terms competence, interkinetic nuclear migration (INM), cell birth, and exit. To facilitate discussion of the model below, we emphasize that birth and exit are viewed as separate activities that are temporally coupled. Birth is the new transcriptional program activated in a differentiating cell that defines its identity, and exit is the cessation of division.

Distance from Notch Predicts Birth

As already described, RPC nuclei undergo INM. Live imaging studies in zebrafish have indicated that nuclei that migrate deeper into the basal layer are more likely to generate a differentiating RTC when they return to the apical surface and undergo mitosis. These data raise the possibility that to achieve birth, nuclei must escape from a diffusable inhibitory signal. An attractive candidate is the antineurogenic factor Notch (Figure 5). Activated Notch is cleaved, releasing its intracellular domain (Nicd), which binds recombination signal-binding protein (Rbpj, also called Cbf1, Suh, Igkrb, Kbf2). Rbpj activates expression of hairy and enhancer of split (Hes) proteins, which are basic helix-loop-helix (bHLH) transcriptional repressors that block expression of proneurogenic genes. In line with the speculative model in Figure 5, Notch is more abundant at the apical side, but there is conflicting literature on whether the diffusable cleaved Nicd fragment or its gene targets (e.g., the Hes proteins) are more abundant/ active in apically located nuclei. The Baier/Link groups found that antibodies to Nicd labeled apically located fish RPCs. Using live imaging, this group also found that a fluorescent protein expressed from the Nicd-inducible her4 promoter was more abundant in RPCs with more apically located nuclei. However, the Reh group finds that Nicd is lowest in the apically located nuclei in the mouse retina, and both the Reh group and the Kageyama group report higher levels of Hes1 in progenitors when their nuclei are located at the basal side of the neuroepithelium in mouse and chick retina and mouse cortex (using highly destabilized Hes1 reporters). Given the highly transient nature of Notch signaling, it will be important to use highly destabilized reporters, like those used in cortex

by Kageyama’s group, for future studies in retina, to better characterize cell-cycle-dependent changes in Notch signaling. Genetic studies prove that Notch is antineurogenic, so it will be important to deduce exactly how it achieves this end.

Genetic analysis in fish shows that, whether or not Nicd/Hes gradients are involved, INM plays a key role in determining birth. In dynactin-1 (mok) mutants, it was found that RPC nuclei migrated deeper (thus appearing to escape Notch signaling) and generated more earlyborn ganglion cells. The mechanisms that control INM distance are unclear, but both actinand microtubulebased motors have been implicated. Notably, RPCs are in S-phase when they approach the basal side and neurogenic bHLH genes are induced in S-phase. Moreover, transplant experiments indicate that both cortical and retinal progenitors can change fate during this period.

Together, these data build a model in which nuclei destined to undergo birth are moved a sufficient distance from the apical surface to escape influence of the Notch pathway, thus permitting induction of neurogenic factors required to activate the birth program.

Birth Does Not Require Exit

Birth and cell-cycle exit are intimately linked, so it is tempting to conclude they must be interdependent (birth needs exit). There is also a model proposing that slowing cell-cycle rate in the last division might also be critical to facilitating birth. Below, we summarize evidence for and against these issues.

In support of the notion that rate reduction favors birth, progenitor cell-cycle length increases with the

gradual transition to symmetric production of two RTCs. In zebra fish, the average cell-cycle length does not predict birth, but, of two sibling RPCs, the one with the longer cycle is more likely to differentiate following M- phase. Moreover, amounts of the Cdk-inhibitor drug olomoucine that slow, but do not stop, division are sufficient to trigger premature neurogenesis in the telencephalon. In support of the idea that birth requires exit, the two are temporally coupled and p27Kip1 induction occurs in the last G2 just prior to RTC birth. Moreover, overexpression of this CKI induces early birth.

The above data are not totally conclusive. Correlations do not distinguish consequence from cause. A longer final cycle in sibling RPCs might reflect deeper migration of the neurogenic partner trying to escape Notch. Olomoucine has targets other than Cdks and importantly, mutations in E2f1, cyclin D1, or Vsx2 that lengthen cell cycle by specific genetic means do not cause a switch to early-born cell types. Thus, whether lengthening the cell cycle is necessary for birth remains moot.

What about exit, is it required for birth? Notchpathway defects trigger both birth and exit, but this is also a correlation that does not distinguish whether they are interdependent or can be uncoupled. Genetic evidence supports the latter since neurogenesis in the retina, forebrain, cerebellum, and inner ear goes ahead in the absence of Rb and differentiating neurons divide ectopically. As discussed earlier, a subset of ectopically dividing neurons eventually undergo apoptosis, while others survive and exit independent of Rb, and these are the source of sporadic retinoblastoma. However, clearly, birth occurs

despite these downstream defects. As with Rb loss, there is also not a shift to late-born cells in retinas lacking p27Kip1

and/or p19Ink4. The interphotoreceptor retinoid-binding protein (IRBP) promoter is activated just before photoreceptors are born, yet its use to overexpress cyclin D1 or E2f1 does not prevent initiation of the birth program, and the resulting photoreceptors divide ectopically. CKI overexpression assays suggest that exit can drive birth, yet do not prove that this is the physiologically relevant or necessary route. Indeed, arresting division with hydroxyurea or aphidicolin in Xenopus embryos does not disrupt most central nervous system (CNS) differentiation, indicating that arrest per se is not neurogenic. Perhaps, CKIs induce early birth by affecting a process other than exit, and, indeed, a mutated version of p27Kip1 incapable of binding and inhibiting Cdks induces neurogenesis through interaction and stabilization of neurogenin 2 (Neurog2 or Ngn2). Alternatively, active cell-cycle components may maintain expression and/or activity of Notch-pathway components and downregulation of the cell cycle would thus block Notch signaling, an intriguing possibility given that E2f regulates the expression of Hes family members.

In summary, while birth and exit are closely coupled, exit is not necessary for birth. They are both induced

following escape from Notch-pathway signals, but in an apparently parallel rather than interdependent fashion.

Mechanisms Linking Birth to Exit

Despite functional independence, birth and exit are coupled temporally, which makes sense given that a single pathway, Notch, is so central in coordinating them both. It is well established that Notch downregulation leads to induction of neurogenic transcription factors through alleviation of Hes1-mediated repression. But how is Notch

connected to cell-cycle exit? Hes1 also represses expression of p27Kip1 mRNA, but in mouse retina p27Kip1 protein

induction during cell birth is regulated at a posttranscriptional level. There are many ways in which p27Kip1 trans-

lation and stability are regulated, yet the specific approach used in the retina is unclear. Activated Notch can induce Skp2 through the same mechanism it uses to activate Hes1 expression and, as noted earlier, Skp2 promotes p27kip1 ubiquitin-mediated degradation (Figure 2). Forkhead transcription factors can promote p27Kip1 stability by downregulating a proteosome subunit. Notch loss down-

regulates Vsx2 and Tlx4, and both are required for high cyclin D1 and low p27Kip1 levels, although the details are

unclear. The intracellular cleaved domain of Notch can directly activate the cyclin D1 promoter, so this mechanism might be employed in the retina. Cyclin D1 downregulation likely ensures activation of Rb protein in newborn neurons. p19Ink4d induction would also facilitate this event, but – although it is regulated at both transcriptional and posttranscriptional levels – the mechanisms used in retinal cells are unclear. Once Rb is activated, it shuts down E2f, and the most critical target is E2f1 since removing the latter rescues all ectopic division and apoptosis in the Rb-null retina.

Conclusion

Birth and exit are coordinately regulated through escape from Notch signaling, which may involve a distancedependent mechanism that relies on polarity signals and INM. It is unclear exactly how the decision to evade Notch is made. Despite their temporal proximity birth is not dependent on exit. The factors that activate the new transcriptional program can be induced independent of signals necessary to shut off division and neurons attempt to develop as they divide ectopically. Failure to couple birth to exit can lead to retinoblastoma, underscoring the importance of maintaining tight coupling. To avoid this catastrophe, some neurons undergo apoptosis, but others adopt Rb-independent means of exiting, which provides a window of time during which neoplastic clones may evolve. Well-known cell-cycle regulators are downregulated and activated to ensure exit parallels birth, and many

of these events are posttranscriptional. Exactly how evading Notch triggers these events in the retina remains to be resolved, although there are clues from work in other cell types.

Acknowledgments

We are grateful to Valerie Wallace, Tom Reh, and Brian Link for comments. Research on retinal development in the Bremner lab is funded by the Canadian Institute for Health Research (CIHR) and the Foundation Fighting Blindness. M. Pacal is supported by a Vision Science Research Program fellowship from the University of Toronto.

See also: Embryology and Early Patterning; Eye Field Transcription Factors; Histogenesis: Cell Fate: Signaling Factors; Intraretinal Circuit Formation; Photoreceptor Development: Early Steps/Fate; Retinal Histogenesis.

Further Reading

Baye, L. M. and Link, B. A. (2008). Nuclear migration during retinal development. Brain Research 1192: 29–36.

Besson, A., Dowdy, S. F., and Roberts, J. M. (2008). CDK inhibitors: Cell cycle regulators and beyond. Developmental Cell 14: 159–169.

Burkhart, D. L. and Sage, J. (2008). Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nature Reviews. Cancer 8: 671–682.

Buttitta, L. A. and Edgar, B. A. (2007). Mechanisms controlling cell cycle exit upon terminal differentiation. Current Opinion in Cell Biology 19: 697–704.

Cayouette, M., Barres, B. A., and Raff, M. (2003). Importance of intrinsic mechanisms in cell fate decisions in the developing rat retina. Neuron 40: 897–904.

Cayouette, M., Poggi, L., and Harris, W. A. (2006). Lineage in the vertebrate retina. Trends in Neuroscience 29: 563–570.

Chen, D., Opavsky, R., Pacal, M., et al. (2007). Rb-mediated neuronal differentiation through cell-cycle-independent regulation of E2f3a.

PLoS Biology 5: e179.

Del Bene, F., Wehman, A. M., Link, B. A., and Baier, H. (2008). Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient. Cell 134: 1055–1065.

Farkas, L. M. and Huttner, W. B. (2008). The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development. Current Opinion in Cell Biology 20: 707–715.

Gotz, M. and Huttner, W. B. (2005). The cell biology of neurogenesis.

Nature Reviews. Molecular Cell Biology 6: 777–788. Kageyama, R., Ohtsuka, T., Shimojo, H., and Imayoshi, I. (2008b).

Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition. Nature Neuroscience 11: 1247–1251.

Levine, E. M. and Green, E. S. (2004). Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors. Seminars in Cell and Developmental Biology 15: 63–74.

Malumbres, M. and Barbacid, M. (2009). Cell cycle, CDKs and cancer: A changing paradigm. Nature Reviews. Cancer 9: 153–166.

Nelson, B. R., Hartman, B. H., Georgi, S. A., Lan, M. S., and Reh, T. A. (2007). Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells. Developmental Biology 304: 479–498.

Pacal, M. and Bremner, R. (2006). Insights from animal models on the origins and progression of retinoblastoma. Current Molecular Medicine 6: 759–781.

Shimojo, H., Ohtsuka, T., and Kageyama, R. (2008). Oscillations in notch signaling regulate aintenance of neural progenitors. Neuron 58: 52–64.

van den Heuvel, S. and Dyson, N. J. (2008). Conserved functions

of the pRB and E2F families. Nature Reviews. Molecular Cell Biology

9: 713–724.

Wallace, V. A. (2008). Proliferative and cell fate effects of Hedgehog signaling in the vertebrate retina. Brain Research 1192: 61–75.

Glossary

Angiogenesis – The formation of new blood vessels from preexisting ones, generally by sprouting. Central retinal artery – A branch of the ophthalmic artery that enters the eye via the optic nerve. Choriocapillaris – An exceptionally dense capillary bed that nourishes the posterior choroid up to the level of the equator of the eye.

Circle of Zinn – An annular artery surrounding the optic nerve. Its branches contribute to the pial circulation, the optic nerve at the level of the lamina cribrosa and to the nerve fiber layer of the optic disk. Fenestrae – The circular openings in the choriocapillaris facing Bruch’s membrane that

˚

measure approximately 800 A. The fenestrae of the choriocapillaries have a diaphragm covering.

Posterior ciliary arteries – The branches of the ophthalmic artery that form the blood supply to the choroid.

Vasculogenesis – The formation of new blood vessels through de novo formation of new endothelial cells.

Vortex veins – The venous collecting vessels draining the choroid, ciliary body, and iris.

Choroidal Vascular Network

Embryology

At the fourth week of gestation in humans (5-mm stage), the undifferentiated mesoderm surrounding the optic cup begins to differentiate and form endothelial cells adjacent to the retinal pigment epithelium (RPE). These early vessels are the precursors of the choriocapillaris, which ultimately envelop the exterior surface of the optic cup. Concurrently, the hyaloid artery branches from the primitive dorsal ophthalmic artery and passes along the embryonic (choroidal) fissure to enter the optic cup. Shortly thereafter, the condensation of the mesoderm occurs at the site of future choroidal and scleral stroma with the gradual onset of pigmentation in the outer neuroepeithelial layer of the optic cup, the RPE. By the fifth week of development (8–10-mm stage), the RPE becomes more melanized and the vascular plexus extends along the entire exterior surface of the cup from the posterior pole to the optic cup rim.

By the sixth week (12–17-mm stage), the choriocapillary network begins to develop a basal lamina. This, together with the basement membrane of the RPE, forms the initial boundaries of Bruch’s membrane separating the neural retina from the choroid. Concomitantly, rudimentary vortex veins develop in all four quadrants of the eye. Except for the two basal lamina (of the RPE and choriocapillaris), the only other component of Bruch’s membrane at this stage is a collagenous central core. The choroidal capillary network becomes almost completely organized by the eighth week (25–30-mm stage) with connections to the short posterior ciliary arteries. By the eleventh week (50–60-mm stage), the posterior ciliary arteries show extensive branching throughout the choroid. It is only following the third and fourth months of gestation that most of the choroidal vasculature matures.

While the molecular mechanisms regulating choroidal development have not been fully defined, it is largely accepted that the presence of differentiated RPE and its secretion of growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF-9), are critical for the physiological development and differentiation of the choroidal vascular network.

Gross Anatomy

Almost the entire blood supply of the eye comes from the choroidal vessels, which originate from the ophthalmic arteries. The left and right ophthalmic arteries arise as the first major branch of the internal carotid, usually where the latter break through the dura to exit the cavernous sinus. In some individuals (around 10%), the ophthalmic artery arises within the cavernous sinus, while in others (around 4%), it arises from the middle meningeal artery, a branch of the external carotid.

The ophthalmic artery shows a wide variation in the branching pattern as it approaches the eye. The posterior ciliary arteries, which form the blood supply to the choroid, and the central retinal artery, which enters the eye via the optic nerve, are branches of the ophthalmic artery (Figure 1). Other branches of the ophthalmic artery supply the lacrimal gland, extraocular muscles, and lids.

The choroid (also referred to as the posterior uveal tract) is vascularized by two separate arterial systems:

(1) the short posterior ciliary arteries, which supply the posterior choroid and (2) the long posterior ciliary arteries, which supply the anterior portion of the choroid (as well as the iris and ciliary body).

Approximately 16–20 short posterior ciliary arteries penetrate the sclera in a circular pattern surrounding the optic nerve, with the distance between these vessels and the nasal side of the nerve being closer than that on the temporal side. These arteries anastomose within the sclera to form the circle of Zinn, an annular artery surrounding the optic nerve (Figures 1 and 2). The branches from the circle of Zinn contribute to the pial circulation, the optic nerve at the level of the lamina cribrosa, and the nerve fiber layer of the optic disk. Other branches from the circle of Zinn along with direct branches from the short posterior ciliary arteries enter the choroid to provide the arterial blood supply to the posterior uveal track. These arteries divide rapidly to terminate in the choriocapillaris, an exceptionally dense capillary bed that nourishes the posterior choroid up to the level of the equator of the eye.

The two long posterior ciliary arteries penetrate the sclera on either side of the optic nerve near the level of the horizontal meridian of the eye. The temporal long posterior ciliary artery enters the sclera approximately 3.9 mm from the temporal border of the optic nerve while the nasal long posterior ciliary artery enters approximately 3.4 mm from the nasal border of the optic nerve. Additional long posterior ciliary arteries are present that course farther toward the anterior segment before penetrating the sclera, usually more anterior than the entry of the temporal and nasal long posterior ciliary arteries. The long posterior ciliary

arteries course through the suprachoroid, begin to branch just anterior to the equator, and contribute to the circulation of the iris and ciliary body. Just anterior to the equator, some branches of these vessels course down into the choroid and branch to terminate in the choriocapillaris from the ora serrata back to the equator of the eye.

In general, the larger diameter arteries of the choroid are found most proximal to the sclera, in an area referred to as the lamina fusca. These arteries continue to branch and ultimately form the extensive choriocapillaris adjacent to the acellular Bruch’s membrane located on the basal side of the RPE (Figure 3). The capillary network of the choriocapillaris is approximately 3–18 mm in diameter and oval shaped in the posterior eye, becoming gradually wider and longer as it moves toward the equatorial region (approximately 6–36 mm wide by 36–400 mm long). The network becomes irregular in the peripheral third of the choroid owing to the entrance and exit of arterioles and venules. Compared to capillaries in other organs, the choriocapillary lumen are significantly larger, with a diameter of nearly 20 mm in the macular region and 18–50 mm in the periphery. A network of collagen fibrils surrounds the choriocapillaris and provides a structural supportive framework.

From the choriocapillaris, venous collecting vessels emerge that ultimately exit the eye through the vortex veins (Figure 1). In addition to the choroid, the vortex veins also drain the ciliary body and iris circulation. The number of vortex veins is variable, with at least one per

eye quadrant; however, the total number per eye is usually seven, with more found on the nasal side than are found temporally. The vortex veins usually exit the sclera at the equator or up to 6 mm posterior to this location after forming an ampulla near the internal sclera. The venous

branches that open into the anterior and posterior regions of the vortex venular network are oriented along the meridian and are mostly straight, while those joining on the lateral and medial sides have a circular orientation. The vortex veins, in turn, empty into the superior and

Superficial capillary plexus

Deep capillary plexus

Choriocapillaris

Choroid

Henle’s fiber layer

Fovea

Photoreceptor layer

Pigmented epithelium

Bruch’s membrane

Sclera

inferior ophthalmic veins, which leave the orbit and enter the cavernous sinus.

Physiology

The primary function of the choroid vasculature is to provide nutrients and oxygen to the outer retina. The loss of the choriocapillaris, as occurs in central areolar choroidal sclerosis, results in atrophy of the overlying retina. The choriocapillary network is unique in that it lies in a single plane below Bruch’s membrane. Normal choroidal circulation occurs when both choroidal arterial and venous pressures are above 15–20 mm of Hg, which is the normal physiological intraocular pressure. In addition, since the blood flow rate through the choroid is relatively high compared to other tissues and is regulated by the arterial vessel diameter, it causes relatively lower amounts of oxygen to be extracted from each milliliter of blood.

The wall of the choriocapillaris facing Bruch’s membrane is fenestrated with circular openings (fenestrae)

˚

measuring approximately 800 A. The fenestrae of the choriocapillaries are unique in that they have a diaphragm covering them, unlike those seen in the renal glomerulus. These fenestrae allow easy movement of large macromolecules into the extracapillary compartment. Fluid and macromolecules escaping from these leaky vessels percolate through Bruch’s membrane and have access to the basal side of the RPE. The passive movement of fluid and macromolecules from this leaky circulation is blocked from reaching the subretinal space (interphotoreceptor matrix) by zonula occludens junctions that form a continuous belt-like barrier near the apical border of the RPE. Thus, the RPE is able to block the passive movement of the large molecules and fluid from the choroid, allowing the RPE to function as the outer portion of the blood retinal barrier.

Pathology

The exudative (wet form) of age-related macular degeneration (AMD) is characterized by an abnormal proliferation of the choriocapillaris with occasional invasion through Bruch’s membrane into the subretinal space. Leakage of the fluid and/or blood often leads to retinal or RPE detachment and loss of central vision. VEGF, basic fibroblast growth factor (bFGF), and pigment epithelial-derived growth factor (PEDF) have been postulated to play a role in the development of choroidal neovascularization (CNV). Some studies have suggested that the thickening of Bruch’s membrane that occurs with age and in AMD could result in decreased permeability. The survival factors for the choriocapillaris, such as VEGF, which are secreted basally from the RPE, could remain sequestered in Bruch’s membrane and be unable to reach the endothelial cells of the choriocapillaris.

This could potentially lead to atrophy of the choriocapillaris that is seen in dry AMD and may be the initiating event for local hypoxia, and upregulation of VEGF and CNV. Myopia is the second most common cause of CNV. Presumed ocular histoplasmosis syndrome, multiple white dot syndrome, multifocal choroiditis, punctate inner choroidopathy, birdshot chorioretinopathy, and the healing phase of choroidal ruptures are the other causes of CNV.

Hyaloid Vascular Network

Embryology

During the first gestational month, the posterior compartment of the globe contains the primary vitreous comprised of a fibrillary meshwork of ectodermal origin and vascular structures of mesodermal origin. At the 5-mm stage, the primitive dorsal ophthalmic artery sprouts off the hyaloid artery, which passes through the embryonic choroidal fissure and branches within the cavity of the primary optic vesicle. Behind the lens vesicle, some of the branches make contact with the posterior side of the developing lens (capsula perilenticularis fibrosa), while others follow the margin of the cup and form anastomoses with confluent sinuses to form an annular vessel. The arborization of the hyaloid artery forms a dense capillary network around the posterior lens capsule (tunica vasculosa lentis, TVL) and surrounding the lens equator. In addition, capillary branches are given off that course throughout the vitreous (vasa hyaloidea propria). The capsulopupillary vessels anastomose with the annular vessel around the rim of the optic cup and connect to the choroidal vasculature through which venous drainage occurs. By the sixth week (17–18-mm embryo), the annular vessel sends loops forward and centrally over the anterior lens surface. By the end of the third month, the anterior portion of the TVL is replaced by the pupillary membrane, which is supplied via loops from the branches of the long posterior ciliary arteries and the major arterial circle. The development of the hyaloid vasculature is almost complete at the ninth week (40 mm) stage. The venous drainage from the vessels of the anterior lens capsule and, subsequently, from the pupillary membrane and from the capsulopupillary vessels occurs through vessels that assemble into a network in the region where the ciliary body will eventually form and will anastomose and feed into the venules of the choroid.

Normally, the hyaloid vascular system begins to regress during the second month of gestation. This process begins with atrophy of the vasa hyaloidea propria, followed by the capillaries of the TVL, and, finally, the hyaloid artery (by the end of the third month). The occlusion of the regressing capillaries by macrophages appears to be a critical step for atrophy to occur. As the vascular structures regress, the primary vitreous retracts, and collagen fibers and a ground substance of hyaluronic acid are produced, forming a

secondary vitreous. By the fifth to the sixth month of gestation, the posterior compartment is primarily composed of the secondary vitreous, and the primary vitreous is reduced to a small, central structure, the Cloquet canal, which is a thin, S-shaped structure that extends from the disk to the posterior surface of the lens.

Physiology

The hyaloid vascular system apparently supplies the nutritive requirements of both the lens and the developing retina before the acquisition of the retinal vasculature. The localization of VEGF in the TVL and papillary membrane may be responsible for fenestrae being present only in the hyaloid capillaries facing the lens. The hypoxia-inducible factor and VEGF have been postulated to play a role in the development and regression of the hyaloid. Other factors that have a part to play in regression are Wnt receptor (Lrp5), Frizzled-4, collagen-18, Arf, Ang2, and BMP-4.

Pathology

Persistent hyperplastic primary vitreous occurs owing to the failure of the hyaloid vasculature to completely regress. The TVL, the anterior extension of the primary vitreous, is comprised of a layer of vascular channels originating from the hyaloid artery, the vasa hyaloidea propria, and the anterior ciliary vessels. The anterior part of this system is supplied by the ciliary system and the posterior part by the hyaloid artery and its branches. The posterior system usually regresses completely by the seventh month of gestation, while the anterior part follows by the eighth month. Two clinical forms of persistent hyperplastic primary vitreous have been identified based on the vascular system that fails to regress: (1) persistent TVL that mainly affects the anterior segment and is now termed anterior hyperplastic primary vitreous and

(2) posterior hyperplastic primary vitreous. The hallmark of posterior hyperplastic primary vitreous is the presence of a retinal fold. The lens is usually clear, but may form a cataract over time if the membranous vessels grow forward to enter the lens through the posterior capsule. Other complications include secondary angle-closure glaucoma, microophthalmia, vitreous membranes, tractional retinal detachment at the posterior pole, and a hypoplastic (underdeveloped) or dysplastic (disorganized) optic nerve head. Patients with the anterior hyperplastic primary vitreous often have the best prognosis for visual recovery. The posterior pole is usually normal, with no evidence of a retinal fold or abnormalities in the optic nerve or macula. Patients characteristically present with the appearance of a whitish mass behind the lens (leukokoria) early in life. Occasionally, elongated ciliary processes are present and the eye is microphthalmic. Intralenticular hemorrhage can occur if the fibrovascular membrane invades the lens. Other complications

include secondary angle-closure glaucoma, strabismus, and coloboma iridis.

Retinal Vascular Network

Embryology

Until the fourth month of gestation, the retina remains avascular as the hyaloid vasculature provides the nutrients to the developing retina. At the fourth month (100-mm stage), the primitive vascular mesenchyme cells near the hyaloid artery invade the nerve fiber layer. At later stages in development, the hyaloid artery regresses back to this point, which marks the posterior origin of the retinal circulation. In the fourth month of gestation, the first retinal vessels appear when solid endothelial cords sprout from the optic nerve head to form a primitive central retinal arterial system. By the sixth month, these vessels start developing a faint lumen that occasionally contains a red blood cell. At this time, the vessels extend 1–2 mm from the optic disk and continue to migrate outward extending to the ora serrata nasally and to the equator temporally by the seventh to eighth month. Pericytes or mural cells are conspicuously absent from the vessels at this time and do not appear until 2 months after birth. The retinal vasculature achieves the adult pattern by the fifth month after birth. The relative roles of angiogenesis versus vasculogenesis in the development of the retinal vascular network are still controversial. However, it is generally accepted that VEGF secreted in a temporal and spatial pattern by microglia and astrocytes plays a critical role in the development of the superficial and deep layers of the retinal vasculature. Platelet-derived growth factor (PDGF) produced by the neuronal cells induces the proliferation and differentiation of astrocytes, which respond to the hypoxic environment by secreting VEGF. These cells establish a gradient of VEGF and a track for endothelial cells to follow. The hypoxia-inducible factor has been shown to be critical in the hypoxia-induced regulation of retinal vascular development. VEGF isoforms have been shown to perform highly specific functions during developmental retinal neovascularization.

Gross Anatomy

Usually, the only arterial blood supply to the inner retina is from the central retinal artery that runs along the inferior margin of the optic nerve sheath and enters the eye at the level of the optic nerve head (Figure 2). Within the optic nerve, the artery divides to form two major trunks and each of these divides again to form the superior nasal and temporal and the inferior nasal and temporal arteries that supply the four quadrants of the retina. The retinal venous branches are distributed in a relatively similar pattern. The major arterial and venous branches and the successive divisions of the retinal vasculature are present in the