Claus Cursiefen

VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.

Lymphangiogenesis, an important initial step in tumor metastasis and transplant sensitization, is mediated by the action of VEGF-C and -D on VEGFR3. In contrast, VEGF-A binds VEGFR1 and VEGFR2 and is an essential hemangiogenic factor. We re-evaluated the potential role of VEGF-A in lymphangiogenesis using a novel model in which both lymphangiogenesis and hemangiogenesis are induced in the normally avascular cornea. Administration of VEGF Trap, a receptor-based fusion protein that binds and neutralizes VEGF-A but not VEGF-C or -D, completely inhibited both hemangiogenesis and the outgrowth of LYVE-1(+) lymphatic vessels following injury. Furthermore, both lymphangiogenesis and hemangiogenesis were significantly reduced in mice transgenic for VEGF-A(164/164) or VEGF-A(188/188) (each of which expresses only one of the three principle VEGF-A isoforms). Because VEGF-A is chemotactic for macrophages and we demonstrate here that macrophages in inflamed corneas release lymphangiogenic VEGF-C/VEGF-D, we evaluated the possibility that macrophage recruitment plays a role in VEGF-A-mediated lymphangiogenesis. Either systemic depletion of all bone marrow-derived cells (by irradiation) or local depletion of macrophages in the cornea (using clodronate liposomes) prior to injury significantly inhibited both hemangiogenesis and lymphangiogenesis. We conclude that VEGF-A recruitment of monocytes/macrophages plays a crucial role in inducing inflammatory neovascularization by supplying/amplifying signals essential for pathological hemangiogenesis and lymphangiogenesis.

Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages

In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.

Descemet Membrane Endothelial Keratoplasty Versus Descemet Stripping Automated Endothelial Keratoplasty

PURPOSE To evaluate visual outcome and endothelial cell survival after Descemet membrane endothelial keratoplasty (DMEK) in comparison with Descemet stripping automated endothelial keratoplasty (DSAEK). DESIGN Single-center, retrospective, consecutive case series. METHODS Thirty-eight eyes of 38 consecutive patients undergoing DMEK, who completed a 6-month follow-up, were compared with 35 eyes of 35 consecutive patients undergoing DSAEK for Fuchs endothelial dystrophy or pseudophakic bullous keratopathy. Main outcome measures included best-corrected visual acuity (in logarithm of the minimal angle of resolution [logMAR] units) and endothelial cell density within a 6-month follow-up. RESULTS Best-corrected visual acuity increased from 0.70 ± 0.48 logMAR and 0.75 ± 0.32 logMAR before surgery to 0.21 ± 0.14 logMAR and 0.48 ± 0.19 logMAR 3 months after DMEK and DSAEK (P < .001), respectively, and to 0.17 ± 0.12 logMAR and 0.36 ± 0.15 logMAR 6 months after DMEK and DSAEK (P < .001), respectively. Endothelial cell density decreased from 2575 ± 260 cells/mm(2) and 2502 ± 220 cells/mm(2) before surgery to 1498 ± 244 cells/mm(2) and 1778 ± 420 cells/mm(2) 3 months after DMEK and DSAEK (P < .001), respectively, and to 1520 ± 299 cells/mm(2) and 1532 ± 495 cells/mm(2) 6 months after DMEK and DSAEK (P = .483), respectively. Central corneal thickness decreased from 652 ± 92 μm before surgery to 517 ± 45 μm 6 months after DMEK, and from 698 ± 137 μm before surgery to 618 ± 66 μm 6 months after DSAEK. CONCLUSIONS DMEK provided faster and more complete visual rehabilitation when compared with DSAEK. However, there were no significant differences concerning endothelial cell survival within a 6-month follow-up.

A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.

Purpose: Lamellar techniques for selective replacement of diseased corneal structures have recently been improved. Descemet membrane endothelial keratoplasty (DMEK) allows the sole replacement of the endothelium-Descemet membrane layer (EDM). However, widespread use of DMEK is currently limited because of problems with donor preparation namely the tearing of the Descemet membrane and the difficulty to unfold the EDM graft in the anterior chamber (AC). Methods: A standardized DMEK procedure that allows safe preparation of EDM, atraumatic introduction of EDM into the AC, reliable orientation of EDM during surgery, and stepwise unfolding within the AC is described in 80 patients. Visual acuity and corneal endothelial cell density were assessed. Results: A stepwise approach using a novel bimanual underwater technique to harvest EDM from donor corneal buttons allows reproducible generation of grafts without tearing the Descemet membrane. Injection of the EDM roll into the AC is achieved by use of a standard injector cartridge, whereas the depth of AC is maintained by an irrigation handpiece. Marks at the margin of EDM allow orientation. Finally, unfolding EDM in the AC is achieved by sequential use of water jets and air bubbles. In the early phase of the learning curve, 4 patients were regrafted because of graft failure. Endothelial cell density decreased from 2600 ± 252 to 1526 ± 341 cells per square millimeter 1 month after DMEK. Conclusions: A novel technique for graft preparation and EDM injection results in improved safety with a high rate of successful DMEKs.

Corneal Higher-Order Aberrations after Descemet's Membrane Endothelial Keratoplasty

PURPOSE We compared corneal higher-order aberrations (HOAs) in eyes after Descemets membrane endothelial keratoplasty (DMEK), Descemets stripping automated endothelial keratoplasty (DSAEK), and penetrating keratoplasty (PK), and in a control group that had not undergone surgery. DESIGN Retrospective analysis of clinical data. PARTICIPANTS Thirty eyes of 30 patients who had undergone standard DMEK, 20 eyes of 20 patients after DSAEK, 20 eyes of 20 patients after PK, and 20 eyes of 20 controls were analyzed. METHODS In addition to standard postoperative examinations, each participant was analyzed with the Pentacam high-resolution rotating Scheimpflug imaging system (Pentacam HR, Oculus, Wetzlar, Germany). Data were compared between groups. MAIN OUTCOME MEASURES Visual acuity and HOAs. RESULTS The mean follow-up was 6.5 ± 1.2 months after DMEK, 22.6 ± 11.8 months after DSAEK, and 103.1 ± 74.2 months after PK. There were no statistically significant differences for the anterior 4.0-mm zones between the DMEK group and the controls or between the DMEK and DSAEK groups. The DMEK procedure compared with PK showed statistically significant differences in all terms for the 4.0-mm zones. All combined Zernike terms for mean posterior aberrations of the central 4.0-mm zones showed statistically significant higher aberrations for DMEK compared with controls. The DMEK procedure compared with DSAEK showed statistically significant lower mean values for all combined Zernike terms, except for coma and coma-like terms in the central 4.0-mm zones of the posterior corneal surface. Compared with PK, DMEK showed statistically significant lower mean values for all combined Zernike terms for the central 4.0-mm zones of the posterior corneal surface, except for spherical aberration (SA) and SA-like terms. Best spectacle-corrected visual acuity (BSCVA) after DMEK was statistically significantly better than after DSAEK (P=0.001) and PK (P=0.005). There was no statistically significant difference when BSCVA was compared with controls (P=0.998). CONCLUSIONS Both DSAEK and PK exhibit increased posterior corneal HOAs even years after surgery. Patients receiving DMEK display only slight changes in posterior corneal HOAs.

Bacterial keratitis early after corneal crosslinking with riboflavin and ultraviolet-A

We report a case of bacterial keratitis 3 days after corneal crosslinking for keratoconus. The patient complained of increasing pain and redness combined with blurred vision in the treated eye starting on the first postoperative day. Clinical examination showed multiple stromal infiltrations and moderate anterior chamber inflammation. Corneal scraping revealed an Escherichia coli infection, which was successfully treated with fortified tobramycin and cephazolin eyedrops for several weeks. This is the first report of a case of rare postoperative complication resulted in an avascularized corneal scar and permanent reduction of the visual acuity.

Corneal Neovascularization as a Risk Factor for Graft Failure and Rejection after Keratoplasty: An Evidence-Based Meta-analysis

TOPIC Preoperative corneal neovascularization (CNV) is thought to be associated with an increased rate of corneal graft failure and potentially also graft rejection. CLINICAL RELEVANCE New therapeutic options that offer differential influence on the ingrowths or regression of either corneal blood or lymphatic vessels force us to re-evaluate the known data about the role of CNV in keratoplasty. METHODS Electronic databases and corneal registries were searched (up through September 2008). Results were reported both descriptively for each study and using random effects meta-analysis. Potential moderating factors for the association between vascularization and graft failure and rejection were examined using metaregression analysis. RESULTS Nineteen studies reporting on a total of 24,944 grafts undergoing keratoplasty were included. An increase in the risk of graft failure and rejection in the presence of pathologic CNV was seen in studies with a pooled risk ratio of 1.32 (95% confidence interval [CI], 1.15-1.49) for graft failure and 2.07 (95% CI, 0.98-3.15) for graft rejection. There was evidence of incremental increase of risk for graft failure and rejection as more corneal quadrants were affected by neovascularization. The 2 factors predictive of increased risk of neovascularization and graft failure were increased recipient age (P = 0.003) and male gender (P = 0.046). CONCLUSIONS Graft failure and rejection risk increase with an increasing number of corneal quadrants affected by neovascularization before keratoplasty. These data support the study of novel topical antiangiogenic therapies at the cornea to precondition such a cornea for future corneal grafting. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Immunohistochemical localization of vascular endothelial growth factor, transforming growth factor alpha, and transforming growth factor beta1 in human corneas with neovascularization.

Purpose. To analyze presence and distribution of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)&agr;, and TGF&bgr;1 in human corneas with neovascularization due to different corneal diseases. Methods. Indirect immunohistochemistry for VEGF, TGF&agr;, and TGF&bgr;1 was performed on paraffin-embedded corneas obtained by keratoplasty. Corneas from each of the four main groups of histopathologic diagnoses associated with corneal neovascularization were analyzed (scarring after keratitis, graft rejection/insufficiency, acute necrotizing keratitis, scarring after mechanical/chemical injury). Subclassification of inflammatory infiltrates was done using immunohistochemistry for CD3 (T-lymphocytes) and CD68 (macrophages). Results. The analyzed angiogenic factors were detectable in corneas from all four histopathologic groups in a similar distribution; capillary endothelial cells, stromal and intravascular inflammatory cells (T-lymphocytes, macrophages), and basal corneal epithelial cells stained positive for the tested angiogenic factors. Conclusion. The angiogenic factors VEGF, TGF&agr;, and TGF&bgr;1 are detectable in human corneas with neovascularization. Their distribution is quite uniform in different corneal diseases, resulting in corneal angiogenesis. An antiangiogenic therapy inhibiting corneal neovascularization by antagonizing angiogenic factors would have to counteract several angiogenic factors.

Optimizing Descemet Membrane Endothelial Keratoplasty Using Intraoperative Optical Coherence Tomography

IMPORTANCE Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. OBJECTIVE To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. DESIGN Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. SETTING Department of Ophthalmology, University of Cologne. PARTICIPANTS Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. EXPOSURE Descemet membrane endothelial keratoplasty. MAIN OUTCOMES AND MEASURES Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. RESULTS Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling (R2 = 0.5 [P = .006]). CONCLUSIONS AND RELEVANCE Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Promotion of Graft Survival by Vascular Endothelial Growth Factor A Neutralization After High-Risk Corneal Transplantation

OBJECTIVE To evaluate whether hemangiogenesis, lymphangiogenesis, and concomitant invasion of mononuclear phagocytes occurring after high-risk corneal transplantation in already vascularized high-risk recipient corneal beds increase the risk for subsequent immune rejection. METHODS Three intrastromal sutures were left in place for 6 weeks in the corneas of BALB/c mice, causing neovascularization. Three weeks after suture removal, keratoplasty was performed (donors C57BL/6 mice). The treatment group received a vascular endothelial growth factor A (VEGF-A)-neutralizing cytokine trap at 0, 4, 7, and 14 days postoperatively (Fc protein was used as the control treatment). Morphometry was performed in corneal flat mounts using lymphatic endothelial hyaluronan receptor-1 (a specific lymphatic endothelial marker), CD31 (a panendothelial marker), and F4/80 (a marker for mononuclear phagocytes). RESULTS After corneal transplantation, significant additional hemangiogenesis (mean area covered by vessels [SD], 68% [18%] postoperatively vs 40% [18%] preoperatively; P = .03) and lymphangiogenesis (12% [1.3%] postoperatively vs 9% [2.8%] preoperatively; P = .03) were observed. Postoperative neutralization of VEGF-A inhibited operation-induced hemangiogenesis (35% [8%]; P = .007) and lymphangiogenesis (6% [1.6%]; P = .03) and decreased the recruitment of mononuclear phagocytes into the graft (mean [SD], 501 cells/mm(2) [152] in treated mice vs 684 cells/mm(2) [35] in Fc controls; P = .03). After 8 weeks, 23% of the treated corneas were not rejected, whereas all control corneas were rejected after 21 days (P = .007). CONCLUSIONS Neutralization of VEGF-A after high-risk corneal transplantation effectively inhibits postoperative hemangiogenesis, lymphangiogenesis, and recruitment of antigen-presenting cells and improves corneal graft survival. CLINICAL RELEVANCE Blocking of VEGF-A after high-risk corneal transplantation may be a novel approach to improve graft survival.