Corneal neovascularization is a pathological condition characterized by the abnormal growth of blood vessels into the normally avascular cornea. This significant ophthalmologic condition not only disrupts corneal transparency but also poses a threat to visual acuity, potentially leading to blindness if untreated.1 Intertwined with corneal NV is lipid keratopathy. Together, they significantly impact corneal transparency. Lipid keratopathy is characterized by the deposition of lipids within the corneal stroma, frequently following the development of neovascularization.2

Slit-lamp photograph showing corneal neovascularization extending into the central cornea with secondary intrastromal lipid leakage into the region of the visual axis. Also evident are anterior blepharitis and secondary bulbar conjunctival injection.

Understanding the intricacies of corneal neovascularization requires a multifaceted approach that encompasses the pathogenesis, clinical manifestations, advanced clinical imaging techniques and contemporary treatment strategies. Here, we’ll delve into the cellular and molecular mechanisms of corneal neovascularization, including the role of angiogenic factors and inflammatory cytokines,3,4 and explore the pathological processes underlying corneal neovascularization. We’ll also explain how this exploration is crucial for the development of targeted therapies aimed at modulating these pathways.

 

Cellular and Molecular Mechanisms in CN

There are several factors that contribute to corneal neovascularization:

• Angiogenic factors. Angiogenic factors produced by inflammation make up a key pathological process in the development of corneal NV. Corneal NV involves the activation of vascular endothelial cells (VECs) in response to hypoxia or inflammation. One of the key angiogenic factors is Vascular Endothelial Growth Factor.5 VEGF promotes endothelial cell proliferation, migration and new vessel formation. The VEGF/VEGFR pathway is one of the most critical, with VEGF binding to its receptors on endothelial cells to activate downstream signaling cascades. VEGF exerts its effects primarily through three separate tyrosine kinase cell-surface VEGF receptors (VEGFR1-3), with VEGF-A, VEGFR-1 (Fms-like tyrosine kinase 1, Flt-1) and VEGFR-2 (kinase insert domain protein receptor, KDR/(fetal liver kinase 1, Flk-1) being strongly expressed in inflamed and vascularized human corneas and, thus, may play an important role in corneal NV.5 In addition to VEGF-A and VEGFR2, other VEGF peptides play significant roles in NV. Aside from important functions in the NV of VEGF-A and VEGFR2, the activation of VEGFR1 leads to the loss of pericytes. One study showed that angiogenic defects caused by pericyte depletion are phenocopied by intraocular injection of VEGF-A or pericyte-specific inactivation of the murine gene encoding VEGFR1.6

VEGF is an important mediator of angiogenic signaling in inflammation to promote endothelial cell survival, proliferation and migration. It’s upregulated in response to hypoxic cornea via the Hypoxia-inducible factor-1 (HIF-1) pathway.7 Hypoxia-inducible factors (HIFs) are crucial regulators of angiogenesis in response to low oxygen levels. HIF-1α, in particular, is stabilized under hypoxic conditions and translocates to the nucleus, where it induces the transcription of VEGF and other proangiogenic genes. The interplay between HIF-1α and VEGF is a key driver of angiogenesis under hypoxic conditions in corneal NV. A study strongly implicated corneal HIF-1α as a component of the inflammatory and neovascular cascade initiated by hypoxia and further suggested that HIF-1α was a proximal regulator of VEGF expression in a mouse model of closed eye contact lens wear.7

Basic fibroblast growth factor (bFGF) is another potent angiogenic factor implicated in corneal NV. The bFGF stimulates endothelial cell division and migration by binding to fibroblast growth factor receptors (FGFRs), which activates downstream signaling pathways such as the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. A mathematical model validated FGF- and VEGF-induced MAPK signaling and phosphorylation of ERK, which promotes cell proliferation. Signaling is initiated by FGF binding to the FGFR1 and heparan sulfate glycosaminoglycans (HSGAGs) or VEGF binding to VEGFR2 to promote downstream signaling. 8

Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, are involved in the degradation of the extracellular matrix (ECM), facilitating angiogenesis.9,10 During the formation of corneal NV, vascular endothelial cells traverse the basement membrane and ECM to enter the tissue. MMP-2 and MMP-9 bind to type-1 and 4 collagens, gelatins and laminin to degrade denatured collagens and gelatins and interfere with the proper functioning of the corneal epithelial barrier.

In a mouse model, the levels of MMP-2 and MMP-9 were elevated in the epithelial cells of neovascularized vessels in the corneal epithelium and stroma.11 The presence of MMP-2 and MMP-9 was found before NV formation, and their levels rose alongside the accumulation of inflammatory cells and NV. This suggested that MMP-2 and MMP-9 have an important role in the production of corneal NV.11,12

Several signaling pathways are involved in the regulation of corneal NV. The PI3K/Akt signaling pathway inhibits angiogenesis in malignant liver tumors, melanoma, hemangioma and renal cell carcinoma by regulating vascular endothelial remodeling. 13–15 The PI3K/Akt signaling pathway is an important downstream pathway of the STAT3 protein. This pathway is critical in the regulation of NV through VEGF.16,17 A study showed that the suppression of the PI3K/Akt signaling pathway, which is controlled by STAT3, effectively inhibits corneal NV.18 The Notch signaling pathway also plays a significant role in angiogenesis. A bFGF-induced mouse study showed that the notch signaling pathway regulates VEGF expression to affect corneal angiogenesis.19 The NF-κB signaling pathway is another important regulator of corneal NV. NF-κB is activated in response to various stimuli, including proinflammatory cytokines, and promotes the transcription of genes such as VEGF to promote corneal NV progression. 20

• Antiangiogenic factors. The cornea maintains its avascularity through the action of antiangiogenic factors, which counterbalance the effects of angiogenic stimuli. Pigment epithelium-derived factor (PEDF), a 50 kDa glycoprotein, is an important antiangiogenic factor, which inhibits endothelial cell migration and tube formation. Under normal physiological conditions, there’s a dynamic balance between VEGF and PEDF, which prevents NV.21 PEDF suppresses VEGF signaling angiogenesis and is used as a viable therapeutic agent for the treatment of corneal and other ocular NV.22,23

Corneal NV is closely linked to inflammation in the cornea, primarily caused by an imbalance between angiogenic and antiangiogenic elements. Tissue inhibitors of metalloproteinases (TIMPs) counteract MMP activity, thereby inhibiting new vessel formation. A study showed that TIMP3 binds to VEGFR-2 to suppress VEGF’s binding and inhibit downstream signaling and angiogenesis.24

Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, is an antiangiogenic protein that inhibits endothelial cell proliferation and migration by interacting with receptor such as CD36. A mouse study showed that TSP-1 ligated with CD36 on monocytic cells inhibited VEGF. 25

Endostatin, a proteolytic fragment of collagen XVIII, also plays a critical role in inhibiting angiogenesis by blocking the pro-angiogenic activity of VEGF and bFGF. Endostatin and its derivatives significantly inhibited vascular endothelial cell proliferation in vitro and suppressed corneal NV in vivo.26

• Cytokines. Inflammatory cytokines play a crucial role in the initiation and progression of corneal NV. Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that promotes angiogenesis by upregulating VEGF and bFGF expression in corneal epithelial cells and fibroblasts. A study has reported that TNF-α promotes hemangiogenesis in the mouse trachea under inflammatory conditions.27 In a wild type mice study, TNF-α enhanced VEGF and inducible nitric oxide synthase (iNOS) expression by peritoneal macrophage whereas the intraocular mRNA expression of angiogenic factors, including VEGF, iNOS was retarded severely in TNF receptor 1-deficient mice.28

TNF-α is a pivotal pro-inflammatory cytokine involved in CNV. It’s primarily produced by macrophages, T-cells, and corneal epithelial cells in response to injury or infection. TNF-α exerts its effects by binding to its receptors to trigger downstream signaling cascades that promote angiogenesis. In a TNF transgenic mice study, TNF stimulated VEGF-C expression and increased nuclear factor kappa B (NF-κB) binding to an NF-κB sequence in the VEGF-C promoter.29 NF-κB is a transcription factor that regulates the expression of various genes involved in inflammation and angiogenesis, including VEGF and IL-1β. The nuclear translocation of NF-κB related to the inflammatory response is inhibited by regulatory protein IkappaB (IκB), which prevents nuclear translocation of NF-κB by arresting it in the cytoplasm.30 Upon TNF-α binding, the IκB kinase complex is activated, leading to the phosphorylation and degradation of IκB, an inhibitor of NF-κB. This allows NF-κB to translocate to the nucleus and induce the expression of pro-angiogenic genes.31

Interleukin-1 (IL-1) is another key mediator that enhances the production of pro-angiogenic factors. An IL-1 receptor antagonist knockout mice study showed corneal NV with increased intracorneal macrophage infiltration and increased expression of VEGF and iNOS. 32 IL-1 also enhances the production of other inflammatory cytokines, amplifying the inflammatory response and promoting angiogenesis.

Conversely, anti-inflammatory cytokines such as interleukin-10 (IL-10) can suppress angiogenesis by downregulating the expression of proangiogenic growth factors. The multifunctional cytokine interleukin (IL)-10 is mostly known for its anti-inflammatory and regulatory effects on the immune response. Several studies have shown that IL-10 has antiangiogenic properties. 33,34 IL-10 promoted the production of antiangiogenic TIMPs 35 and augmented angiogenesis in IL-10-deficient mice that was accompanied by upregulation of proangiogenic MMP-2 and -9.36 However, IL-10 has also been shown as a proangiogenic molecule in a murine model of inflammation-associated choroidal NV37 and in the suture model for corneal NV.38

Left: Slit-lamp photograph displaying a significant amount of lipid keratopathy involving the central and mid-peripheral cornea. Also seen is corneal neovascularization. Right: Corneal neovascularization is clearly seen against this red reflex photograph. Photo: Charles Bouchard, MD.

Lipid Keratopathy

LK can be classified as idiopathic or it can be secondary, resulting from ocular infection, inflammation or trauma. Idiopathic LK can be identified by the presence of neutral fat, glycoproteins, cholesterol and lipid deposits in the stromal layer of the cornea and the adjacent limbus.39 The accumulation of lipids in idiopathic LK may result from an overproduction of lipids or a deficiency in fat metabolism. Typically, idiopathic LK manifests bilaterally and doesn’t have a history of serum lipid problems or corneal NV. In contrast, secondary LK commonly arises from the accumulation of lipids in a cornea NV. As a result, any condition that causes corneal NV might potentially trigger LK.

Corneal NV can cause the accumulation of lipids and subsequently lead to LK through many mechanisms. During the formation of new blood arteries, increased amounts of lipoproteins can be transported to these regions, particularly when there are elevated levels of serum lipoproteins. Because there’s less pericyte coverage, less basement membrane layering and fewer tight junctions in these newly formed vascular tissues, they are highly porous, which permits lipid and cholesterol leakage.40 Fibroblasts endocytose this cholesterol-rich lipid, which is subsequently transported to lysosomes where it’s deposited in droplets in the cytoplasm. Over time, these fibroblasts get encircled by lipid, resulting in cell necrosis and the accumulation of crystalline material in the corneal stroma.41 Necrosis also triggers an inflammatory reaction, which worsens the process of NV and promotes the accumulation of lipids. Lipid deposition can produce NV as the condition worsens, and NV in turn can promote further lipid deposition, which further reduces visual acuity.

 

Diagnosis

Patients with corneal NV often present with symptoms such as decreased visual acuity, photophobia and ocular discomfort. Accurate diagnosis of NV and lipid keratopathy relies on a combination of clinical examination and advanced imaging techniques. Slit lamp photography, in vivo confocal microscopy (IVCM), angiography, and optical coherence tomography angiography are the main diagnostic techniques used in clinic to assess the corneal surface.

On slit-lamp examination, corneal NV appears as fine, tufted blood vessels extending from the limbus into the corneal stroma. In lipid keratopathy, the cornea may exhibit yellowish-white opacities corresponding to lipid deposits, often in association with vascularized corneal scars. Slit-lamp biomicroscopy remains the primary tool for initial assessment, allowing for detailed visualization of the corneal structures and identification of NV and lipid deposits. While artificial intelligence algorithms have somewhat improved the diagnostic capabilities of slit lamp photography, the enhancement in diagnosability remains limited.

New imaging modalities have shown to be beneficial as adjunct methods. IVCM obtains sequential images across different corneal layers and offers insights into cellular changes within the corneal stroma. 42 It can visualize the presence of corneal vessels by detecting chemicals that are absorbed by the permeable vessels. Lipid deposits in LK may also be characterized with the use of IVCM. It frequently detects crystalline formations inside the stroma in cases of both idiopathic and secondary LK.43 IVCM is a non-invasive method for imaging the cornea, while angiography relies on the use of particular dye agents. Fluorescein angiography can be used to assess the patency and leakage of corneal vessels.44 Both fluorescein and indocyanine green leaking can indicate neovascular development and aid in the diagnosis. Fluorescein angiography and indocyanine green angiography facilitate early detection and precise mapping of NV.45 They are reliable measures of the level of advancement of corneal NV with evaluation of the duration and direction of blood flow, even when corneal scars are present.46

 Optical coherence tomography is now widely used for studying arteries because of the recent improvements in image capture and algorithms. OCT angiography has emerged as a new technique that enables the viewing of blood flow dynamics without the need for injecting contrast agents. Continuous imaging enables the visualization of ocular vessel blood flow through non-invasive OCTA, thereby avoiding the adverse reactions associated with invasive angiography.47 The existing integrated software for OCTA enables users to perform sequential scans to create a three-dimensional assessment of the lesion and its related blood vessels.48 This is valuable for determining the stage of the disease and devising treatment strategies. However, OCTA is unable to distinguish vascular activity and blood flow direction when compared to fluorescein angiography and indocyanine green angiography despite its advancements. AS-OCTA is another promising diagnostic tool for detecting abnormal blood vessel growth in the cornea. AS-OCT provides high-resolution cross-sectional images of the cornea, enabling precise measurement of corneal thickness and the extent of NV.49 However, the scanning range of AS-OCTA is narrower compared to standard angiography procedures, requiring numerous successive scans to cover all corneal quadrants. While the acquisition speed is still poor despite the short scanning duration, involuntary eye movement might also impair clarity while assessing corneal NV.50 As non-invasive AS-OCTA continues to advance, it’s expected to replace invasive ocular examination methods for diagnosing corneal NV. This will allow for faster, more accurate and more efficient diagnosis.

 

Therapeutic Implications

Understanding the molecular mechanisms underlying corneal NV and LK has significant therapeutic implications. Anti-VEGF therapies, such as bevacizumab, ranibizumab and aflibercept have been developed to inhibit VEGF signaling and are used to treat corneal NV, secondary LK and other angiogenic ocular diseases.51,52 Similarly, inhibitors of bFGF and other proangiogenic factors are being explored as potential treatments. Therapies aimed at enhancing the activity of TSP-1, endostatin or PEDF could help to restore the balance between angiogenic and antiangiogenic signals and inhibit pathological NV. Anti-inflammatory therapies targeting cytokines like TNF-α and IL-1β could be beneficial in reducing the inflammatory component of corneal NV. Drugs that inhibit NF-κB signaling or other proinflammatory pathways may help to decrease the expression of angiogenic factors and mitigate NV. However, most treatment options have resulted in a similar lack of long-term success. Although the primary approach to corneal NV is the treatment of the underlying infectious and inflammatory stimulus, topical corticosteroids have demonstrated little efficacy in reversing corneal vascularization.53 Corneal transplantation alternatives, such as penetrating keratoplasty, have a significant likelihood of graft failure and a high risk of rejection in eyes with corneal NV.

Although the occurrence of corneal NV is increasing, there is still a lack of effective therapeutic techniques. Argon laser therapy has been suggested to induce vascular occlusion by focusing a focused beam of light into the abnormal blood vessels.54 However, laser-induced tissue damage can exacerbate NV and hence LK by triggering inflammation and the release of angiogenic molecules. Fine needle diathermy (FND) was adopted by cauterization and elimination of the abnormal vessel. Nevertheless, complications may arise, such as temporary whitening of the cornea, bleeding inside the corneal stroma, recanalization or the development of collateral vessels.55 Other vasodestructive treatments, such as photodynamic therapy, cautery, and suture ligation, have unsatisfactory long-term outcomes.

An optimal therapy should eradicate existing aberrant corneal NV and inhibit future vascularization. MICE, mitomycin-C (MMC) intravascular chemoembolization, is a newly proposed surgical method for addressing corneal NV and LK.56 MMC inhibits the proliferation of vascular endothelial cells, hence impeding the development and regenerative potential of blood vessels.57 Studies have reported MICE successfully treated corneal NV with LK,56 and corneal vascularization prior58 or after failed keratoplasty.59

In MICE, a 1.0 cc syringe is filled with MMC (0.4 mg/mL), which is then connected to a 33-gauge needle. The physician identifies the greatest lumen of a corneal vessel located just inside the limbus. Then the needle is positioned and inserted into the vessel at an approximate angle of 15 degrees relative to the corneal surface. A little amount of MMC (0.01 to 0.05 ml), with a maximum volume not exceeding 0.05 ml, is injected into the efferent and afferent vessels. The ocular surface is thoroughly irrigated using balanced salt solution to eliminate any remaining ocular surface MMC. One drop of moxifloxacin 0.5% and prednisolone acetate 1% is administered at the conclusion of the procedure.

Given the technical difficulty of this procedure, surgeons can confirm the intravascular injection of MMC by observing the blanching of blood vessels during the injection. If the needle’s entire bevel isn’t inserted through the stroma, the MMC will follow the path of least resistance, which is the ocular surface, rather than entering the vessel. In contrast, the administration of MMC into the anterior chamber could have catastrophic consequences, the surgeon must prevent the complete penetration of the full-thickness cornea. Some patients can achieve clinical success with one injection, but some may need a second MICE treatment to resolve the high-velocity blood vessels. It could be because the blood arteries were very deep and didn’t receive effective chemoembolization, or MMC has entered the intrastromal space rather than the blood vessel. Surgeons can monitor the vessels blanching during the procedure. Particular attention should be paid to the needle’s appropriate angling, which should be approximately 15 degrees, in order to successfully cannulate the vessel. When the injection is correctly positioned and angled, a tiny amount of MMC (0.01-0.05 ml) is sufficient to fill both the efferent and afferent vessels.

The MICE therapy resulted in remission of the corneal NV and later partial absorption of the LK in a reported case series. 56 Around one to three weeks after treatment, lipid and blood accumulated inside the corneal stroma which was absorbed within one to two months, resulting in a feathery appearance of the remaining lipid with decreased hyper-reflectivity on AS-OCT. The absorbed lipid may flatten the cornea, causing induced astigmatism. However, the corneal flattening and induced astigmatism were improved, and the visual acuity remained stable.

High dose (>0.05 ml of 0.4 mg/ml MMC) or injecting MMC toward the limbus may cause potential systemic adverse effects such as abdominal pain, nausea and vomiting. We suggest thoroughly irrigating the surface of the eye with balanced salt solution after the procedure to minimize the amount of MMC that comes into contact with the eye’s surface. In the case of small vessels, the technical aspects of MICE can be particularly challenging. Consequently, it’s advised to determine the biggest vessel. The hydrostatic pressure from the injection will fill the whole network with MMC, including the small vessels if properly placed.

In conclusion, corneal NV is a complex pathological process involving a delicate balance between pro-angiogenic and antiangiogenic factors, regulated by a network of cytokines and molecular pathways. VEGF, bFGF, MMPs are drivers of angiogenesis, counteracted by antiangiogenic factors like PEDF, TIMPs, TSP-1 and endostatin. The interplay of cytokines further modulates this balance. Key signaling pathways, including VEGF/VEGFR, Notch, HIF-1α, and NF-κB, coordinate the cellular responses leading to corneal NV. Understanding these mechanisms provides a foundation for developing targeted therapies to treat and prevent this vision-threatening condition.

Although the indications and procedure for MICE are undergoing changes, MICE is increasingly becoming one of the potential primary methods for treating corneal NV and LK. Performing MICE therapy is technically challenging, and large comparison studies with longer follow-up periods are warranted to evaluate its safety and effectiveness.

 

Victor Wang is an undergraduate at the University of Central Florida, Orlando, Florida. He majors in biomedical sciences. 

David Yang is a research associate in the Ophthalmology Department at Specialty Retina Center/ Advanced Research, LLC, Florida.

Dr. Cheng is a Resident at Broward Health, Department of Ophthalmology, Florida, and a voluntary clinical associate professor at Florida International University, Department of Ophthalmology in Miami.

Dr. John is in private practice in the Chicago area, and is a clinical associate professor at Loyola University at Chicago.

Financial Disclosure: No authors have any proprietary interest in this study.

Funding/Support: This study was not supported by any grant.

Conflict of Interest: None of the authors have conflicts of interest.

Corresponding Author: Thomas John, MD

 

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