Chapter 3 of 4

Neovascularization

The corneal angiogenic (and lymphangiogenic) privilege

The cornea is avascular, and this lack of vessels — blood and lymphatic — is essential to its transparency, refractive function and immune privilege. This "angiogenic privilege" is not passive: it results from an active balance between pro- and anti-angiogenic factors ^[7].

Endogenous inhibitors

Three complementary strategies keep the cornea free of vessels:

Sequester VEGF before it can act. The soluble receptor sVEGFR-1 (sFlt-1), expressed by the corneal epithelium, captures circulating VEGF and prevents it from reaching endothelial cells — its loss is enough to trigger vessel growth, which established that corneal avascularity directly depends on sVEGFR-1 ^[8].
Block vascular endothelial cells directly. Angiostatin, endostatin, thrombospondins and IPAS (inhibitory PAS domain protein, a truncated splice variant of the transcription factor HIF-3α that acts as a dominant-negative inhibitor of the hypoxic response ^[17]) act downstream: they promote endothelial-cell apoptosis or inhibit the integrins needed for their migration. PEDF (pigment epithelium-derived factor) is among the most potent endogenous angiogenesis inhibitors known ^[15].
Repeat the same logic on the lymphatic side. A soluble receptor VEGFR-3 (sVEGFR-3) sequesters VEGF-C and shuts down lymphangiogenesis, securing corneal a-lymphaticity ^[9,16] — a mechanism as decisive as the blood side, since it is the lymphatic route that lets the recipient's immune system "see" the graft and become sensitized to it.

Transparency = maintained avascularity

Optical clarity rests on a permanent angiogenic brake. While the balance tips anti-angiogenic, neither blood nor lymphatic vessels colonize the stroma — which also shields the cornea from the immune system.

Corneal neovascularization

Various diseases break this balance and trigger neovascularization: infectious keratitis, chemical burns, inflammatory diseases and chronic hypoxia from contact-lens overwear. The response often combines hem-angiogenesis and lymph-angiogenesis, the latter especially harmful to transplant immunity ^[7,9].

The consequences are twofold. Neovascularization compromises transparency, but it also facilitates infiltration by immune and antigen-presenting cells and weakens immune privilege — raising the risk of rejection. Clinically, stromal new vessels evolve as: oedema → haemorrhage → lipid keratopathy and fibrosis.

A proteolytic link ties this chapter to the previous one: MMPs do not only degrade matrix, they also generate angiogenesis inhibitors. Angiostatin (a kringle K1-4 fragment of plasminogen) is produced by proteolytic cleavage, notably by stromelysin-1 (MMP-3); after excimer keratectomy, angiostatin and endostatin co-localize with MMPs, indicating that the latter shape these anti-angiogenic molecules and help maintain avascularity (Gabison et al., 2004). The proteolytic system thus has a dual face: destructive for the stroma (melting), protective of the vascular privilege. The classification of corneal neovascularization mechanisms was framed in this context (Chang, Gabison, Kato & Azar, 2001).

Management targets the cause first (control of inflammation and the surface); targeted options include anti-VEGF and diathermy/photocoagulation of feeder vessels, as part of preparing high-risk grafts.

Two transparencies at stake

Neovascularization costs not only optical transparency: by opening the cornea to immune cells, it undermines its "immunological transparency". Controlling inflammation, hem- and lymph-angiogenesis determines the survival of high-risk grafts ^[9].

Glossary of abbreviations used in this course

Scientific abbreviations and acronyms used throughout the 10 pages of this course, listed alphabetically.

95% CI: 95% confidence interval
AAV: adeno-associated virus (gene-therapy viral vector)
ABCG2: ABCG2 transporter, a limbal stem-cell marker
AMT: amniotic membrane transplantation
anti-VEGF: anti-angiogenic treatment targeting VEGF
ASC: inflammasome adaptor protein (Apoptosis-associated Speck-like protein containing a CARD)
BrdU: bromodeoxyuridine, a cell-proliferation marker
CCL2: CCL2 chemokine, synonym of MCP-1; recruits circulating monocytes
CCR2: receptor for CCL2; marker of newly recruited monocytes/macrophages
CD147: cluster of differentiation 147; synonym of EMMPRIN and basigin
CD163: M2 macrophage marker; scavenger receptor for haemoglobin-haptoglobin
CD206: mannose receptor; marker of M2 polarization
CD74: cell-surface receptor for MIF
CDVA: corrected distance visual acuity
CGRP: calcitonin gene-related peptide
CTGF: connective tissue growth factor
CX3CL1: fractalkine, a chemokine involved in monocyte/macrophage recruitment
CXCR2/CXCR4: chemokine receptors, also receptors for MIF
DAMP: damage-associated molecular pattern
DESI: direct epithelial–stromal interaction
EGF: epidermal growth factor
EGFR: epidermal growth factor receptor
EMMPRIN: extracellular matrix metalloproteinase inducer; synonym of CD147
ETDRS: reference visual-acuity scale used in clinical research (Early Treatment Diabetic Retinopathy Study)
FasL: Fas ligand, an apoptosis-inducing protein
FISH: fluorescence in situ hybridisation
GAG: glycosaminoglycan(s)
GM-CSF: granulocyte-macrophage colony-stimulating factor
GVHD: graft-versus-host disease
HGF: hepatocyte growth factor
HIF-3α: hypoxia-inducible factor, 3-alpha subunit
HMGB1: high mobility group box 1, an alarmin released by injured cells (DAMP)
IFN-γ: interferon gamma
IGF-1: insulin-like growth factor 1
IL-1: interleukin-1
iNOS: inducible nitric oxide synthase
IPAS: inhibitory PAS domain protein
KGF: keratinocyte growth factor
LASIK: laser-assisted in situ keratomileusis
LogMAR: logarithmic visual-acuity scale
LOX: lysyl oxidase, a collagen cross-linking enzyme
LPS: bacterial lipopolysaccharide
M1: classically activated macrophage, pro-inflammatory profile
M2: alternatively activated macrophage, pro-resolving/reparative profile (subtypes M2a-M2d, M2eff)
MEN2B: multiple endocrine neoplasia type 2B
MerTK: receptor tyrosine kinase involved in efferocytosis
MIF: macrophage migration inhibitory factor
MRI: magnetic resonance imaging
NGF: nerve growth factor
NK: neurotrophic keratitis
NLRP3: NLRP3 inflammasome (NOD-, LRP- and pyrin domain-containing protein 3)
NO: nitric oxide
NSAID: nonsteroidal anti-inflammatory drug
PACAP: pituitary adenylate cyclase-activating polypeptide
PAF: platelet-activating factor
PAMP: pathogen-associated molecular pattern
PDGF: platelet-derived growth factor
PED: persistent epithelial defect
PEDF: pigment epithelium-derived factor
PGE₂: prostaglandin E2
PRK: photorefractive keratectomy (laser surface ablation)
pro-NGF: precursor form of NGF
RGTA: matrix-therapy ReGeneraTing Agent, e.g. Cacicol
rhNGF: recombinant human NGF (cenegermin)
ROS: reactive oxygen species
Smad: signalling proteins of the TGF-β pathway
SPK: superficial punctate keratitis
sVEGFR-1: soluble form of VEGF receptor-1 (= sFlt-1)
sVEGFR-3: soluble form of VEGF receptor-3
TGF-β: transforming growth factor beta
TIMP: tissue inhibitor of metalloproteinases
TLR: Toll-like receptor
TNF-α: tumour necrosis factor alpha
TRPV4: transient receptor potential vanilloid 4, a mechanosensitive ion channel
uPA: urokinase-type plasminogen activator
V1: ophthalmic branch of the trigeminal nerve
VEGF: vascular endothelial growth factor
VEGF-C: lymphangiogenic isoform of VEGF
VIP: vasoactive intestinal peptide
YAP/TAZ: Hippo pathway effectors, mechanosensors of matrix stiffness
α-SMA: alpha-smooth muscle actin, a myofibroblast marker