Ângela Carneiro, MD, PhD
Faculty of Medicine of University of Porto, Hospital S. João, Porto, Portugal.




Blood vessels develop and grow by three different basic mechanisms: vasculogenesis in which vessels form by concatenation of vascular precursor cells into solid cords that then lumenize; angiogenesis that is the growth of new blood vessels from pre-existing ones; and intussusception in which new blood vessels form by the proliferation of endothelial cells (EC) that form a pre-existing vessel into the vessel lumen, originating two blood vessels that split into two opposite sides(1).

Angiogenesis, the growth of new vessels from pre-existing ones by sprouting of EC into a previously avascular tissue, is an essential process both in embryonic development and in adulthood(1,2).

It is a complex multistep process involving extracellular matrix degradation and proliferation, survival, migration and anastomosis of EC(2).

The release of extracellular matrix proteases leads to the degradation of blood vessels basal membrane, EC change shape, proliferate, invade stroma and form tubular structures that coalesce.

This requires the coordinated action of a variety of anti and pro-angiogenic factors and cell-adhesion molecules in endothelial cells.

However, if on one side it promotes tissue repair, on the other hand if imbalanced it promotes tissue damage.

If not tightly regulated, the angiogenic process is frequently imbalanced, and associated with several pathological situations(1,3).


Angiogenic mediators and modulation of their expression


Angiogenic process requires the activation of series of receptors by numerous ligands including Placental Growth Factor (PIGF), Fibroblast Growth Factors (FGFs), Angiopoietin-1 and -2 (Ang-1 and -2), Platelet-derived Growth Factor (PDGF), Hepatocyte Growth Factor (HGF), Connective Tissue Growth Factor (CTGF) and Transforming Growth Factors (TGF-α e TGF-β), among many others(1,3-9).

However, there is a consensus that the Vascular Endothelial Growth Factor (VEGF) is the most important angiogenic factor and represents the crucial rate-limiting step during angiogenesis(3,10,11).

VEGF-A is the prototype member of a gene family that also includes placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D, and the orf-virus-encoded VEGF-E(11).

Alternative exon splicing results in the generation of four main VEGF isoforms, which have respectively 121, 165, 189, and 206 amino acids after the signal sequence is cleaved (VEGF121, VEGF165, VEGF189, and VEGF206).

Less frequent splice variants have also been reported, including VEGF145, VEGF183, VEGF162, and VEGF165b(8,11).

VEGF mediates its biological functions in endothelium through binding two highly related receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2.

It is generally agreed that VEGFR-2 is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF-A(3). VEGFR-1 binds not only VEGF-A, but also Placenta Growth Factor (PGF) and fails to mediate a strong mitogenic signal in endothelial cells.

It is now generally agreed that VEGFR-1 plays a role in modulation of activity of VEGF(10). The mediators of angiogenic process can be modulated by some molecules and microenvironmental conditions.

VEGF is upregulated by cyclo-oxygenase (COX-2)(12); inflammation with the hypoxic environment and the cells involved in inflammatory process, release huge amounts of factors that exert effects on EC and degrade the extracellular matrix(2).

Angiogenesis can also be suppressed by inhibitory molecules, such as interferon-α, thrombospondin-1, angiostatin, endostatin or pigment epithelial-derived factor (PEDF)(13-17). It is the balance of stimulators and inhibitors that tightly controls the normally quiescent capillary vasculature.

When this balance is upset pathological angiogenesis develops(18).


Angiogenesis during development of retinal vasculature


During embryogenesis retinal vascularization begins in the most superficial (or inner) retinal layers at the optic nerve head, and radiates outwards from this central point.

It reaches the retinal periphery just before birth(19).

The migration of large numbers of vascular precursor cells (VPCs) from the optic disc is the first event in human retinal vascularization, apparent before 12 weeks gestation(20).

They proliferate and differentiate to form a primordial vascular bed centered on the optic disc. Thus, vasculogenesis is responsible for the formation of the primordial vessels of the inner (superficial) plexus in the central human retina(21).

Formation of retinal vessels via vasculogenesis appears independent of metabolic demand and hypoxia-induced VEGF expression(22).

Angiogenesis is responsible for the formation of the remaining retinal vessels, including increasing vascular density in the central retina, vessel formation in the peripheral retina of the inner plexus, and formation of the outer plexus and the radial peri-papillary capillaries(22).

Formation of the outer plexus begins around the incipient fovea between 25 and 26 weeks of gestation, coincident with the signals indicative of a functional visual pathway and photoreceptor activity(21).

The timing and topography of angiogenesis in the human retina supports the “physiological hypoxia” model of retinal vascular formation, in which angiogenesis is induced by a transient but physiological level of hypoxia as a result of the increased metabolic activity of retinal neurons as they differentiate and become functional(23).


Angiogenesis in retina and choroidal pathologies


Retinal anatomy is highly organized and vascular and avascular compartments are strictly segregated in the retina(1).

The blood-retinal barriers, inner and outer, are fundamental for the integrity of structure and optimization of function in neuro-sensorial retina(24).

Pathological retinal and choroidal angiogenesis generates chaotically orientated and physiologically deficient vessels that do not conform to neuronal histology, which can lead to vision-threatening exudation and haemorrhage(1).

Angiogenesis is a key aspect in many ocular pathologies that are leading causes of blindness in the world, such as neovascular age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity (ROP), central retinal vein occlusion and other diseases associated with ischemia and neovascularization(25).

Although angiogenesis is a highly complex and coordinated process requiring multiple receptors and ligands in endothelial cells, VEGF is a hypoxia-inducible cytokine that appears to be a pivotal element required for the process in a variety of normal and pathological circumstances(3,10).

Vascular endothelial growth factor is a surrogate angiogenic marker, since it acts not only as a mitogen, but also as a survival factor for endothelial cells (EC)(2).

Furthermore, it is also involved in the stimulation of the invasive and migration capacity of EC and in the enhancement of vascular permeability(10).


Angiogenesis and Age-related Macular Degeneration


The diagnosis of age-related macular degeneration rests on signs in the macula, irrespective of visual acuity(26).

The stages of age-related macular degeneration are categorized as either early, in which visual symptoms are inconspicuous, or late usually associated with severe loss of vision(27).

Early AMD is characterized by the presence of drusen and/or hyperpigmentations or small hypopigmentations(26). Late AMD has “dry” and “wet” forms.

However, in the same patient we can find either the dry form in one eye and the wet in the other eye, or the two forms in the same eye. Moreover with time we can see the conversion of wet in dry or dry becoming wet(28).

Age-related changes that predispose to AMD occur in the outer retina, more specifically in the region that includes the photoreceptors, the retinal pigment epithelium (RPE), Bruch’s membrane and the choriocapillaris.

The aging-dependent alterations in the outer retina have been already discussed in another chapter.

AMD-related visual loss is a complex process starting by the deposition of debris in the outer retina(29).

The deposition of insoluble material, the calcification and increase in thickness of Bruch’s membrane, and a less fenestrated and thinner choriocapillaris leads to photoreceptors/retinal pigment epithelium hypoxia resulting in a stimulus for VEGF release(28,30-32).

All the aging changes in outer retina compromise the nutrition of photoreceptors and RPE and create a favourable ambiance for the development of choroidal neovascularization. However other factors – genetic and environmental(33,34) – are also important, but their roles in the development of CNV is discussed in other chapters of this book.

In general terms there are two basic CNV growth patterns, based on the anatomical position of the abnormal vessels with respect to the RPE monolayer, which are related to the Gass classification of choroidal neovascularization(35,36).

Type 1 signifies CNV located in the plane between the RPE and Bruch’s membrane and type 2 neovascularization means that the vessels have penetrated the RPE layer to proliferate in the subneurosensory space(35).

In type 1 growth pattern after breaking through Bruch’s membrane, tufts of CNV extend laterally under the RPE in a horizontal fashion facilitated by the natural cleavage plane between basal laminar deposits and a lipid rich Bruch’s membrane.

This growth pattern recapitulates the choriocapillaris and can provide some nutrients and oxygen to an ischemic RPE/outer retina(36,37).

The type 2 growth pattern occurs usually with one or few ingrowth sites with vascular leakage under the RPE/outer retina, leading to acute visual symptoms(36).

Recently, Yannuzzi proposed a type 3 neovascularization, for retinal angiomatous proliferation (RAP), indicating proliferating vessels within or below the retina itself(38).

This mixed neovascularization, with a presumed dual origin, may have intraretinal neovascularization driven by angiogenic cytokines from Müller cells, endothelial cells, pericytes, and retinal glial cells, and CNV driven by cytokines from the RPE(38).

There is hypothetically neovascularization extending anteriorly from the choroid in conjunction with retinal neovascularization progressing posteriorly, with both circulations destined to anastomose.

The reason that growth patterns vary according to disease and individual may be related to genetic predispositions, environmental mechanisms, variations in composition and anatomy of Bruch’s membrane, cytokine distribution, or other causes(36).

During the dynamic process of development of CNV there is a balance of angiogenesis promoters and inhibitors. In the initiation stage, the RPE and photoreceptors produce VEGF(39).

There is also production by RPE of Interleukin-8 (IL-8) and Monocyte Chemoattractant Protein-1 (MCP), which attract monocytes from the choriocapillaris along the outer surface of Bruch’s membrane(40).

The macrophages tend to concentrate around sites of vascular ingrowth through the Bruch’s membrane and express Tumor Necrosis Factor-α (TNF-α) and Interleukin-1 (IL-1), which up-regulate complement factor-B, activate the complement alternative pathway in the subretinal space, and stimulates RPE cells to produce more VEGF(40,41).

After initiation, CNV grows to a certain size and progresses through the tissue planes by the action of Matrix Metalloproteinases (MMP) produced by EC and macrophages(42).

During this stage of active growth, Angiopoietins (Ang-1 and 2) are expressed, FGFs are produced by RPE and EC, and TGF-β is produced by the RPE(43-45).

CNV stabilizes during the active stage due to a steady state established between MMP and tissue inhibitors of metalloproteinases, Ang-1 and 2, PEDF and VEGF, PDGF and VEGF, plasminogen and fibrin, and others(36,46).

At some point the balance shifts toward antiangiogenic, antiproteolytic and antimigratory activity resulting in the involutional stage of CNV.

When this occurs the angiogenic/proteolitic/migratory cytokine production decreases with a shift toward TGF-β and tissue inhibitors of metalloproteinases production by the RPE(36).

In this involution stage the CNV may become collagenized and form a disciforme scar.


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Last revision: October 2011 by Ângela Carneiro