Vascularized Corneas Research Articles

Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis invivo.

To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels invivo. Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

UV light-mediated corneal crosslinking as (lymph)angioregressive pretreatment to promote graft survival after subsequent high-risk corneal transplantation (CrossCornealVision): protocol for a multicenter, randomized controlled trial

BackgroundGood vision highly depends on the transparency of the cornea, which is the “windscreen” of the eye. In fact, corneal blindness due to transparency loss is the second most common cause of blindness worldwide, and corneal transplantation is the main cure. Importantly, the cornea is normally avascular but can secondarily be invaded by pathological (blood and lymphatic) vessels due to severe inflammation, and the survival prognosis of a corneal graft mainly depends on the preoperative vascular condition of the recipient’s cornea. Whereas transplants placed into avascular recipient beds enjoy long-term survival rates of > 90%, survival rates significantly decrease in pathologically pre-vascularized, so-called high-risk recipients, which account for around 10% of all performed transplants in Germany and > 75% in lower and middle-income countries worldwide.MethodsThis parallel-grouped, open-randomized, multicenter, prospective controlled exploratory investigator-initiated trial (IIT) intends to improve graft survival by preconditioning pathologically vascularized recipient corneas by (lymph)angioregressive treatment before high-risk corneal transplantation. For this purpose, corneal crosslinking (CXL) will be used, which has been shown to potently regress corneal blood and lymphatic vessels. Prior to transplantation, patients will be randomized into 2 groups: (1) CXL (intervention) or (2) no pretreatment (control). CXL will be repeated once if insufficient reduction of corneal neovascularization should be observed. All patients (both groups) will then undergo corneal transplantation. In the intervention group, remaining blood vessels will be additionally regressed using fine needle diathermy (on the day of transplantation). Afterwards, the incidence of graft rejection episodes will be evaluated for 24 months (primary endpoint). Overall graft survival, as well as regression of corneal vessels and/or recurrence, among other factors, will be analyzed (secondary endpoints).DiscussionBased on preclinical and early pilot clinical evidence, we want to test the novel concept of temporary (lymph)angioregressive pretreatment of high-risk eyes by CXL to promote subsequent corneal graft survival. So far, there is no evidence-based approach to reliably improve graft survival in the high-risk corneal transplantation setting available in clinical routine. If successful, this approach will be the first to promote graft survival in high-risk transplants. It will significantly improve vision and quality of life in patients suffering from corneal blindness.Trial registrationClinicalTrials.gov NCT05870566. Registered on 22 May 2023.

On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

The purpose of this study was to examine the role of Elovl3 gene in meibogenesis and the impact of ELOVL3 protein ablation on the physiology of the mouse ocular surface and Meibomian glands (MGs). Elovl3 knockout, ELOVL3-ablated (E3hom) mice and their wild type littermates (E3wt) were studied side by side. E3hom mice had abnormal ocular phenotypes such as delayed eye opening, weeping eyes, crusty eyelids, eyelid edema, highly vascularized cornea and tarsal plates (TPs), slit eye, and increased tearing that resemble symptoms observed in human subjects with various forms of dry eye, MG dysfunction and blepharitis. Lipid profiling of E3hom TPs was conducted using chromatography and mass spectrometry. The analyses revealed that the lipid composition of E3hom TPs was strikingly different from that of their E3wt littermates. The mutation affected major classes of meibomian lipids - cholesteryl esters, wax esters, and cholesteryl esters of (O)-acylated w-hydroxy fatty acids. The studies illuminated the central role of ELOVL3 in producing C21:0-C29:0 fatty acids, including odd-chain and branched ones. Ablation of ELOVL3 leads to selective changes in the lipid composition of meibum, making E3hom mice instrumental in studying the mechanisms of the biosynthesis of meibum and modeling various pathologies of human ocular surface and adnexa.-Butovich, I. A., Wilkerson, A., Bhat, N., McMahon, A., Yuksel, S. On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

Identification of corneal neovascularization-related long noncoding RNAs through microarray analysis.

To reveal the role of long noncoding RNAs (lncRNAs) in corneal neovascularization (CN). We established a murine CN model and performed lncRNA expression profiling to identify differentially expressed lncRNAs between normal and vascularized corneas. Based on Pearson correlation analysis, an lncRNA/mRNA coexpression network was constructed. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of lncRNA-coexpressed mRNAs were conducted to determine the related biological modules and pathological pathways. Real-time polymerase chain reactions were carried out to detect the expression pattern of lncRNA in the clinical samples. A total of 154 differentially expressed lncRNAs were identified between vascularized and normal corneas, including 60 downregulated lncRNAs and 94 upregulated lncRNAs. GO enrichment analysis of lncRNA-coexpressed mRNAs indicated that the biological modules were correlated with extracellular region, DNA binding, and immune response. KEGG pathway analysis indicated that "pathways in cancer" was the most enriched signaling pathway. Moreover, the human ortholog of NR_033585 and lincRNA:chr8:129102060-129109035 reverse strand was found to be differentially expressed between vascularized and avascular corneas. This study provides a novel insight into CN pathogenesis. The intervention of dysregulated lncRNAs may become potential targets for the prevention and treatment of ocular vascular diseases.

Angiopoietin-Like Protein 2 Is a Potent Hemangiogenic and Lymphangiogenic Factor in Corneal Inflammation

We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo. Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1β (IL-1β) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed. Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1β were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas. Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1β expression. Angptl2 may be a novel therapeutic target for preventing blindness.

Histopathological Features in a Case of Peters' Anomaly with Acquired Corneal Staphyloma

We report a case of corneal staphyloma histologically diagnosed as caused by Peters' anomaly. A 62-year-old male had a protruding opaque vascularized cornea that began to bulge from six months ago in the right eye. Since his right eye was blind and he wanted us to remove the eyeball for cosmetic improvement, we enucleated the affected eye. The enucleated tissue was fixed in formalin and embedded in paraffin for histological examination. Hematoxylin and eosin staining showed that the cornea lacked the posterior part of the corneal stroma and Descemet's membrane in the central region and the entire corneal endothelium. The corneal epithelium was keratinized. Collagen type I was strongly positive in peripheral cornea and weakly in protruding stroma. The cells labeled by antibodies against αSMA were scattered in the entire corneal stroma. As judged by the histological findings, the eye with the central corneal staphyloma was diagnosed as Peters' anomaly.

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty.

To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohistochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC (r=0.725, P<0.01). Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.

Comparison of EphA Receptor Tyrosine Kinases and ephrinA Ligand Expression to EphB-ephrinB in Vascularized Corneas

Eph cell surface receptors and their ligands, ephrins, are involved in neuronal patterning and neovascularization. Our purpose is to compare and characterize the expression of ephrinA ligands and EphA receptors to ephrinB ligands and EphB receptors in excised mouse corneal tissue, in corneal epithelial and keratocyte cell lines, and during corneal angiogenesis. Mouse corneal epithelial cells and keratocytes were immortalized using SV40T antigen viral infection of primary cultures. The immortalized epithelial cells and keratocytes were cloned and characterized using antibodies to keratin, vimentin, integrin alpha5beta1, and alpha-smooth muscle actin. Basic fibroblast growth factor pellets were implanted to induce corneal neovascularization. The eyes of wild-type, ephrinB2(tlacZ/+), and EphB4(tlacZ/+) heterozygous mice were harvested and sectioned 7 days after pellet implantation. Confocal immunohistochemistry was performed to compare the expression of the Eph/ephrinA family (EphA1-8, ephrinA1-5) and Eph/ephrinB family (EphB1-4, EphB6 ephrinB1-3). EphA1, EphA3, ephrinA1, ephrinA2, EphB1, EphB4, ephrinB1, and ephrinB2 were detected in wild-type mouse corneal epithelial cells and keratocytes. EphA2 was immunolocalized only in epithelial cells. Also, EphA3, ephrinA1, EphB1, EphB4, and ephrinB1 were immunolocalized to the corneal epithelium and stroma. In the vascularized corneas, ephrinB1 was immunolocalized mainly to the keratocytes around the vessels, and ephrinB2, EphB1, and EphB4 were colocalized mainly with CD31 to the vascular endothelial cells. The characterization of ephrin ligand and Eph receptor expression during cornea angiogensis in this study suggests that the Eph/ephrin family of receptor tyrosine kinases and their ligands may play a role in the regulation of corneal angiogenesis.

Proangiogenic Role of ephrinB1/EphB1 in Basic Fibroblast Growth Factor-Induced Corneal Angiogenesis

Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization.

Long-standing Bullous Keratopathy Is Associated with Peripheral Conjunctivalization and Limbal Deficiency

To investigate whether peripheral corneal neovascularization in bullous keratopathy (BK) is due to conjunctivalization, a sign of limbal stem cell deficiency. Observational case-control study. Sixteen BK patients. Patients were divided into 2 groups: BK without peripheral neovascularization [NV(-) group; 5 patients, 5 eyes] and BK with neovascularization [NV(+) group; 11 patients, 13 eyes]. Evidence of conjunctivalization was evaluated by periodic acid-Schiff staining of impression cytology samples from the peripheral vascularized cornea. The 2 groups' durations of disease also were compared. Penetrating keratoplasty (PK) was performed in all 16 cases, and the 2 groups' durations of reepithelialization after PK were compared. Presence of goblet cells using impression cytology, duration of BK, and duration of postoperative reepithelialization. Goblet cells were found on the peripheral corneal surface in all eyes in the NV(+) group. However, all eyes in the NV(-) group were negative for goblet cells (P<0.0001). Duration of disease was 14.4+/-5.4 months in the NV(-) group and 66.2+/-65.5 months in the NV(+) group (P = 0.030). Duration of postoperative epithelialization was 6.2+/-2.2 days in the NV(-) group and 28.8+/-36.5 days in the NV(+) group (P = 0.046). Conjunctivalization of the peripheral cornea and delayed postoperative epithelialization in BK patients with NV suggest the presence of limbal stem cell deficiency in such patients. Patients with long-standing disease were found to be more prone to neovascularization. For this reason, early surgery may lead to a better surgical outcome.

Endothelin-1 and ETA/ETB Receptor Protein and mRNA

Neovascularization of the cornea causes blindness and increases the risk of immune rejections after keratoplasty. The purpose of this study was to investigate involvement of the potent angiogenic growth factor endothelin (ET)-1 and its receptors, ETA and ETB, in corneal neovascularization. ET-1, ETA, and ETB receptor protein expression was evaluated in nonvascularized and vascularized human corneas by immunohistochemistry. Epithelial ET-1 protein expression of both groups was compared using a semiquantitative scoring system. Double immunofluorescence was used to colocalize ETA and ETB receptor with CD31. In situ hybridization and immunoelectron microscopy analyzed ET-1 and its receptors in normal and vascularized corneas. Nonvascularized corneas displayed ET-1 and ETA/ETB receptor protein and mRNA in epithelial and some corneal endothelial cells. ETA more than ETB receptors were expressed on some keratocytes. In vascularized corneas, ET-1 and ETA/ETB receptor expression was found in the endothelial lining of new blood vessels (as shown by CD31-colocalization). ET-1 protein expression was significantly increased in the epithelium of vascularized corneas (P < 0.001). Immunogold localized ET-1 and its receptors to the nuclear/perinuclear space and to the luminal side of endothelial cells of new blood vessels. In corneal neovascularization, ET-1 protein and mRNA expression is upregulated in epithelial cells. Together with ET-1, ETA, and ETB receptor expression on endothelial cells of ingrown new blood vessels, this points to an involvement of ET-1 and its receptors in corneal angiogenesis. As potent ETA and ETB receptors are available, the endothelin system may represent an additional target for corneal antiangiogenic therapy.

Collagen-Rich Crystalloids in a Scarred Vascularized Cornea

Collagen-rich crystalloids, also referred to as collagenous crystalloids, are uncommon findings in benign salivary gland tumors with myoepithelial differentiation and in cutaneous neoplasms. Herein, we report the presence of collagen-rich crystalloids in the scarred, vascularized cornea of a 56-year-old woman. The patient underwent penetrating keratoplasty, and microscopic examination of hematoxylin-eosin-stained sections disclosed large aggregates of refractile material within the corneal stroma. The deposits were partially birefringent when viewed with polarized light and composed of radially arranged columns (long rectangles) with rounded to pointed tips. The deposits had tinctorial properties of collagen using Masson trichrome and the van Gieson method for collagen, and they stained with Alcian blue, pH 2.5, and Verhoeff elastic stain. They did not stain with Gomori methenamine silver, Snook reticulin stain, or tyrosine using the Baker modification of the Millon reaction. This is the first report, to our knowledge, of collagen-rich crystalloids in the cornea. Their presence in the cornea indicates that these structures may occur in the absence of neoplasia.

Collagen-Rich Crystalloids in a Scarred Vascularized Cornea

Collagen-rich crystalloids, also referred to as collagenous crystalloids, are uncommon findings in benign salivary gland tumors with myoepithelial differentiation and in cutaneous neoplasms. Herein, we report the presence of collagen-rich crystalloids in the scarred, vascularized cornea of a 56-year-old woman. The patient underwent penetrating keratoplasty, and microscopic examination of hematoxylin-eosin-stained sections disclosed large aggregates of refractile material within the corneal stroma. The deposits were partially birefringent when viewed with polarized light and composed of radially arranged columns (long rectangles) with rounded to pointed tips. The deposits had tinctorial properties of collagen using Masson trichrome and the van Gieson method for collagen, and they stained with Alcian blue, pH 2.5, and Verhoeff elastic stain. They did not stain with Gomori methenamine silver, Snook reticulin stain, or tyrosine using the Baker modification of the Millon reaction. This is the first report, to our knowledge, of collagen-rich crystalloids in the cornea. Their presence in the cornea indicates that these structures may occur in the absence of neoplasia.

Symblepharon Associated With Cyanoacrylate Tissue Adhesive

Cyanoacrylate tissue adhesives have been used since 1968 for management of corneal perforations. 1 Symblepharon formation has not previously been associated with cyanoacrylate glue. Symblepharon may be secondary to denuded epithelium, trauma, chemical burns, inflammation from numerous drugs, congenital causes, pemphigus, Stevens-Johnson syndrome, and other exfoliative disorders. 2 We describe a case of symblepharon secondary to chronic inflammation from n -butyl cyanoacrylate tissue adhesive. Report of a Case. —A 43-year-old white woman was involved in an automobile accident and suffered an acid burn to the left eye in June 1989. During the next 8 months, progressive thinning of the scarred vascularized cornea occurred with subsequent perforation in April 1990. n -Butyl cyanoacrylate tissue adhesive (Nexacryl, Tri-Point Medical L.P., Raleigh, NC) was applied to the area. A bandage contact lens was placed over the glue, and the patient was administered topical antibiotic drops. One year later, the tissue adhesive was still

Photodynamic Therapy for Corneal Neovascularization

We have previously described corneal neovascularization (CNV) induced by the intrastromal injection of interleukin-2 (IL-2) in inbred mice. Photodynamic therapy (PDT), administered by a deeply penetrating 630 nm fiberoptic laser, can destroy neoplasms and their associated neovascularization with some selectivity, but can damage neighboring tissues when used for CNV. We performed PDT with a 514 nm ophthalmic argon laser in an attempt to induce regression of CNV and reduce the associated toxicity. Eight weeks following IL-2 injection, mice with CNV were injected i.v. with dihematoporphyrin ether (DHE). Seventy-two hours later, 11 eyes (group I) were irradiated with eight 800 mW, 1000 mu, 2 s spots. Controls included 11 vascularized corneas from mice that received DHE but no laser (group II), 11 that received laser but no DHE (group III), and 35 untreated vascularized corneas (group IV). Comparison of the mean areas of CNV in groups I through IV pretreatment (6.0, 6.5, 6.7, and 7.6 mm2) and 12 weeks posttreatment (4.3, 6.3, 5.6, and 7.5 mm2) revealed that a significant decrease was seen in group I only (p less than 0.04, ANOVA). Complications in group I included blepharitis (9%) and iris damage (18%). Histologic studies revealed no evidence of posterior segment damage. PDT with the 514 nm laser is safe and efficacious for the treatment of IL-2-induced CNV in this model.

The effect of prospective HLA-A and -B matching in 288 penetrating keratoplasties for herpes simplex keratitis.

A retrospective review of 288 penetrating keratoplasties (PKP) for herpes simplex keratitis demonstrated a 60.4% survival rate of clear grafts at 3 years. A highly significant difference in corneal graft survival in severely vascularized corneas (57.1% at 3 years) versus non- or slightly vascularized corneas (82.8% at 3 years) was observed (P = 0.009). Prospectively HLA-A and -B matched grafts in vascularized corneas revealed a significantly improved graft survival when compared to unmatched grafts (P = 0.035). Corneal graft survival in eyes grafted 'à chaud' was not significantly below the graft survival of vascularized corneas with a clinically non-active herpetic corneal disease. Recurrence of herpes simplex in the graft was observed in an earlier postoperative period in HLA-A and -B matched grafts (P = 0.0004).

First experiences with HLA-matched corneal grafts in high risk cases

In highly vascularized corneas the number of graft failures caused by irreversible rejections is higher than in non-or slightly vascularized corneas. The importance of antigen compatibility is demonstrated, especially in these 'high risk' cases. Ten highly vascularized corneas were grafted with HLA-matched donor material; only one reversible rejection was seen in this group. Nine non-or slightly vascularized corneas were grafted with donor material chosen a random. Retrospective HLA matching was performed. Three irreversible rejections were seen in this group. The number of HLA incompatibilities was high.

Interlamellar corneal grafts in rats. Effect of histocompatibility.

A technique of interlamellar keratoplasty was employed to evaluate the influence of difference in histocompatibility on the results of corneal transplantation in highly inbred rats. The degree of corneal clarity and vascularity and the incidence of rejection of relatively conpatible ("weak") and incompatible ("strong") allografts were recorded blindly in recipients with normal and vascularized corneas and in recipients sensitized with donor tissue either before or after corneal grafting. In contrast to the well-known effects of histocompatibility on skin and organ allografts, there was no substantial effect whatsoever of histocompatibility on either the severity of corneal allograft reactions orthe incidence of rejection. Consequently, the results of this experimental investigation suggest that tissue typing may have little or no value in clinical keratoplasty.

Suppression of the corneal homograft reaction by antilymphocyte serum.

In from nine to 14 days, rabbits previously sensitized to their donors' tissues started to reject the rabbit corneal buttons that had been placed in their vascularized corneas. Systemically administered antilymphocyte serum (ALS) significantly retarded or prevented the development of the graft reaction during a 40-day observation period. Animals receiving normal horse serum or ALS systemically developed signs of anaphylaxis and serum sickness after repeated injections.

Suppression of corneal graft reaction by antilymphocyte serum.

Rabbits previously sensitized to rat cornea rejected within six to eight days the rat corneal buttons that had been placed in their vascularized corneas. Goat antirabbit lymphocyte serum (GARLS), administered systemically, significantly retarded or entirely prevented the development of the graft reaction. The subconjunctival administration of GARLS was irritating, and in the doses used it did not significantly alter the graft reaction. The white blood cell and lymphocyte counts dropped dramatically within several minutes after GARLS was injected systemically. Animals receiving normal goat serum systemically developed anaphylaxis and serum sickness after repeated injections, whereas animals receiving GARLS systemically did not show any side effects. Animals receiving GARLS systemically showed diminished numbers of lymphocytes in their thymus glands as well as enlarged and active spleens.