What causes AMD? The mechanisms of pathologic neovascularizations

As the therapy for wet age-related macular degeneration continues to improve, the focus of retinal specialists has turned from not merely destroying or excising neovascular membranes through thermal laser photocoagulation, photodynamic therapy or submacular surgery but to modifying the pathway that leads to wet AMD through drug therapy.

The pathway is in part dependent on angiogenesis, which is in turn crucially dependent on vascular endothelial growth factor (VEGF).^1,2

Angiogenesis

A hallmark of wet AMD is choroidal neovascularization (Figure). Neovascularization is the development of new vessels and can be broadly classified in two ways.

Vasculogenesis is the establishment of a new vessel network from hematopoietic precursor cells that differentiate into endothelial cells. Angiogenesis describes the sprouting of new vessels from an existing vascular network.

The angiogenic process is a complex process, characterized by a cascade of events. It starts within the existing vessels and is associated with changes in endothelial cell activity and intercellular adhesions. Secretion of proteases is necessary for basement membrane degradation, cell migration, and extracellular matrix invasion.

Finally, endothelial cell proliferation and capillary lumen formation are followed by stabilization of the vascular network that involves the reversal of these processes, with re-establishment of the basement membrane, cessation of proliferation, junctional complex formation, and recruitment of pericytes to support the vessel wall.

Pathologic angiogenesis may be both excessive, as is seen in diabetic retinopathy, and insufficient, as in cardiovasculopathy. Ophthalmologists should be aware of this when treating ocular disease by approaching the angiogenic pathway. There may be a risk of serious adverse effects elsewhere in the body.

VEGF

Angiogenesis requires a finely tuned balance between numerous stimulatory and inhibitory signals. Herein, VEGF plays a pivotal role. The term VEGF in this case indicates VEGF-A, also known as vascular permeability factor.

Figure. Confocal microphotograph displaying activated vessels (green). Some cell nuclei (blue) express a proliferation marker (red). Source: Grisanti S.

VEGF-A (hereafter called VEGF) is the best examined representant of the VEGF-platelet-derived growth factor (PDGF) supergene family. This includes also VEGF-B, VEGF-C, VEGF-D, VEGF-E (a virally encoded protein), and placental growth factor (PlGF), all showing varying degrees of homology with VEGF.

Alternative splicing of the VEGF gene results in generation of four major homodimeric polypeptides and other less frequent splice variants. The four dominant VEGF isoforms of 121, 165, 189, and 206 amino acids contain consensus signal sequences for secretion. Depending on their binding domains, these isoforms are more or less soluble.

VEGF has purposes beyond inducing angiogenesis. It is also a strong vascular permeability factor and is important in inflammatory mechanisms. Although ophthalmologists may view VEGF as a largely negative factor, especially regarding CNV, it also has positive effects: It acts as a survival factor for endothelial cells and as neuroprotectant for neurons in the central nervous system and the retina.³

Pathologic angiogenesis

Ophthalmologists know that VEGF is important in angiogenesis and that angiogenesis causes CNV. It is, therefore, logical to state that VEGF has a key role in CNV. However, what is the evidence that VEGF is causative of CNV in patients with neovascular AMD?

Answers can be found in experimental models. In animal experiments, VEGF induces CNV and CNV expresses VEGF.^4-6 If VEGF or VEGF receptors are blocked, then VEGF function and CNV development are inhibited.

Additional suggestions that VEGF is causative of CNV in patients with neovascular AMD can also be derived from the study of human ocular tissue. Within the past decade, several researchers disclosed expression of VEGF within surgically excised CNV.^7,8

In addition to VEGF, however, several other factors have been demonstrated in this pathologic tissue, making it difficult to address one factor as the most important. In recent years, my laboratory examined a large number of CNV membranes excised from patients with neovascular AMD for the expression and presence of VEGF.⁹

Although VEGF can be consistently demonstrated in the CNV membranes in such specimens, it is difficult to determine precisely the temporal relationship of the VEGF expression to the onset of the pathologic angiogenesis and clinical picture.

One way to address this issue was to examine angiogenesis in tissue treated with PDT. Having an artificial “time zero,” the following angiogenic process can be correlated with the clinical appearance of the CNV membrane on fluorescein angiography.

It is known that shortly after PDT (eg, 3 days), fluorescein angiography highlights hypoperfusion of the targeted area.^10-14 This correlates with the histopathologic examination of the excised membrane at this time point, which confirms that most of the vessels are occluded and identifiable only by immunohistochemical markers for endothelial cells.

However, 1 month after PDT, an active leaking CNV membrane will often recur.^14,15 Histopathologic examination of a CNV membrane excised at this timepoint will reveal numerous vessels and an inflammatory infiltrate within the membrane.

A question is whether the clinically and histologically observed revascularization is the result of recanalization of the blood vessels damaged by the PDT or a result of an angiogenic process.

Our studies suggest that it is angiogenesis. After PDT, an increase in proliferating blood vessels within the CNV membrane occurs as evidenced by expression of markers for activated proliferation in the endothelial cells.^{9, 14}

Furthermore, 3 days after PDT when blood vessels are destroyed¹⁶ and/or occluded, retinal pigment epithelial (RPE) cells strongly express VEGF.⁹ In the following, revascularization of the pathologic tissue is associated with additional expression of VEGF by the newly formed vessels and by the inflammatory cellular infiltrate in the stroma.

This angiogenic process may be a result of the insult caused by PDT. Beside occluding the vascular component of the CNV, PDT seems to also affect the choriocapillaris.¹⁷ This can obviously create an ischemic environment. Hypoxia is one of the best promoters of VEGF.

In addition, PDT induces the formation of free radicals, which can also influence and increase VEGF synthesis. As a consequence, upregulated VEGF stimulates the development of new blood vessels responsible for the recurrent CNV membrane.

Inhibition of VEGF

The strong correlation between upregulation of VEGF and revascularization in human tissue supports the idea of a major contribution by this growth factor and the rationale for an anti-VEGF therapy. Concerning not only the pathologic but also the physiologic role of VEGF, the approach has to evaluate a selective inhibitor against a pan-blockade of all isoforms.

The functions covered by the different VEGF isoforms are not fully understood yet. An animal model of retinopathy of prematurity¹shows an uprise in VEGF₁₆₄, (equivalent to human VEGF₁₆₅) that is suggested to be the “pathological” isoform. Blocking VEGF₁₆₄ seems sufficient to block the pathologic correlate of disease with no negative effects on the existing normal, physiologic vessels.

A knockout mouse unable to produce VEGF₁₆₄ is still able to develop normal retinal vessels, suggesting that the VEGF₁₆₄ isoform is important for the disease but not for the development, establishment, and maintenance of normal vessels.¹

Already-established vessels, however, can be influenced by and are dependent on other VEGF isoforms.¹⁸ Blocking all isoforms results in regression of established vessels within 2 days; by 10 days, the regression is more impressive.

VEGF is not only important for new vessel growth and for a trophic effect on established vessels, but it is also important within a pathologic environment.

In an experimental model,¹⁹ researchers induced a retinal ischemia followed by reperfusion. After inhibition of VEGF₁₆₄, survival of the retinal neural cells was similar to that of control cells.

However, when all VEGF isoforms were blocked, more retinal neural cell death occurred, supporting the idea that, physiologically, some VEGF isoforms are important for survival and protection of neural cells.

Turning to anti-VEGF therapy, ophthalmologists need a drug that will effectively inhibit pathologic mechanisms but avoid interference with physiologic mechanisms. —Salvatore Grisanti, MD

Physiologic VEGF expression seems to be present and needed in the eye at all times. RPE cells are one ocular source of VEGF that is secreted in a basal direction toward the choriocapillaris.²⁰

This may explain why the choriocapillaris is a specialized fenestrated vascular network. Flat-mount preparation of the choriocapillaris-RPE complex and analysis of the relationship disclosed an obvious dependency of these structures.²¹ Presence of an RPE was associated with a morphologically healthy vascular network, in the absence of RPE cells; however, choriocapillaris was regressed.

It is to be assumed that physiologic VEGF production by RPE cells is likely responsible for the health of the choriocapillaris. Disease and aging of RPE cells lead to cellular dysfunction and eventually to atrophy and cell death.

In addition, sub-RPE deposits, as seen in early stages of AMD, may function as a barrier. Both components will have a negative impact on VEGF signaling and cause atrophy of choriocapillaris.

Atrophy of this supportive and nutritive structure will in turn have a negative impact on the RPE cells. This may be the point where AMD can turn, depending on whether the damaged RPE cells die, in geographic atrophy. However, other factors such as hypoxia, free radicals, and inflammation may enhance VEGF response.

In addition, proteases secreted by macrophages may disrupt the deposit barrier re-enabling a VEGF signaling and induction of neovascularization from the choroidal bed.

VEGF is a double-edged sword. On one side, it is needed for normal ocular function; on the other, it supports pathologic neovascularization. Turning to anti-VEGF therapy, ophthalmologists need a drug that will effectively inhibit pathologic mechanisms but avoid interference with physiologic mechanisms.

References

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