Cutting edge: lymphatic vessels, not blood vessels, primarily mediate immune rejections after transplantation.

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin alpha5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

Safety profile of topical VEGF neutralization at the cornea.

PURPOSE: Bevacizumab eyedrops inhibit corneal neovascularization. The purpose of this study was to analyze the safety profile of VEGF-A neutralization at the ocular surface. METHODS: Bevacizumab eyedrops (5 mg/mL) and an antimurine VEGF-A antibody (250 microg/mL) were applied to normal murine corneas five times a day for 7 and 14 days. Subsequently, corneas were analyzed for morphologic changes by light and electron microscopy. In a mouse model of corneal epithelial abrasion, the effects of topically applied anti-VEGF antibodies on epithelial wound healing were analyzed: the treatment group received bevacizumab (5 mg/mL) or the antimurine VEGF-A antibody (250 microg/mL) as eyedrops, and the control group received an equal volume of saline solution. After 12, 18, and 24 hours, corneas were photographed in vivo with and without fluorescein staining for morphometry. Afterwards the mice were killed, and eyes were removed for histology, immunohistochemistry with Ki67/DAPI, and electron microscopy. The effect of midterm anti-VEGF therapy on corneal nerve density was assessed by staining corneas treated with an FITC-conjugated anti-neurofilament antibody and morphometric analysis. RESULTS: Murine corneas treated with two different types of anti-VEGF antibody eyedrops did not show obvious corneal morphologic changes at the light and electron microscopic levels. Furthermore, anti-VEGF antibody eyedrops had no significant impact on the wound healing process after corneal epithelial injury or on normal murine corneal nerve fiber density. CONCLUSIONS: Topical neutralization of VEGF-A at the corneal surface does not have significant side effects on normal corneal epithelial wound healing, normal corneal integrity, or normal nerve fiber density. Therefore, anti-VEGF eyedrops seem to be a relatively safe option to treat corneal neovascularization.

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation.

PURPOSE: To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS: Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS: Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS: Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.

Borrelia-associated crystalline keratopathy with intracorneal detection of Borrelia garinii by electron microscopy and polymerase chain reaction.

PURPOSE: First report of a patient with Borrelia-associated crystalline keratopathy with intracorneal evidence of Borrelia garinii by polymerase chain reaction (PCR) and electron microscopy (EM). METHODS: Report of a 67-year-old patient with medical history of recurrent iridocyclitis and arthritis presented with a bilateral, progressive, asymmetric crystalline keratopathy, which was particularly pronounced in the peripheral temporal superior cornea. After penetrating keratoplasty, crystalline keratopathy with stromal haziness recurred. Corneal regrafting was performed. The corneal specimen from the penetrating keratoplasty was examined by light and EM as well as by PCR. RESULTS: In the explanted corneal graft, as well as retrospectively in the corneal specimen from the first keratoplasty, spirochetelike bodies and fragments were detected by light and EM. Borrelia burgdorferi sensu lato DNA was demonstrated by broad-range (16S rDNA) PCR. A more precise identification as Borrelia garinii serotype 5 was possible by analyses of the flaB and ospA gene sequences. Borrelia-specific serological tests showed borderline titers in immunofluorescence and weak reaction in immunoblot, respectively. CONCLUSIONS: This case illustrates that borreliae must be considered as a cause of crystalline keratopathy; Borrelia-specific serological tests can be false negative; explanted cornea specimens of etiologically unclear crystalline keratopathy should be analyzed by EM or PCR for detection of pathogens; and prolonged antibiotic treatment might be effective to prevent progression or recurrence of the disease.

Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation.

PURPOSE: Inflammatory corneal hem- and lymphangiogenesis occurring both prior to as well as after penetrating keratoplasty significantly increase the risk for subsequent immune rejections. The purpose of this study was to analyze whether the blocking anti-VEGFR3 antibody mF4-31C1 is able to inhibit the outgrowth of pathologic new lymphatic vessels in a mouse model of suture-induced, inflammatory corneal neovascularisation, and whether this antibody specifically inhibits lymphangiogenesis without affecting hemangiogenesis. METHODS: Three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6 weeks old) and left in place for 7 days to induce neovascularisation. The treatment group (n = 9) received the anti-VEGFR3 antibody mF4-31C1 intraperitoneally on the day of surgery and 3 days later (0.5 mg/mouse). Control mice received an equal amount of control IgG solution. For immunohistochemistry, corneal flat mounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry was performed with the image analysis software analySIS;B (Soft Imaging Systems GmbH, Münster, Germany). To improve the objectivity and precision of the morphometrical analysis, we established a modified method using grey filter sampling on monochromatic pictures. RESULTS: The mF4-31C1 antibody-treated mice displayed nearly complete inhibition of lymphangiogenesis compared with IgG controls (p < 0.006). In contrast, there was no significant inhibitory effect observed with respect to blood vessel growth (p > 0.05). CONCLUSIONS: Inflammatory corneal lymphangiogenesis seems to depend on VEGFR3-signalling. By blocking this receptor the ingrowths of lymphatic vessels can be inhibited almost completely, and specifically without affecting hemangiogenesis. This may open new treatment options to promote (corneal) graft survival without affecting hemangiogenesis.

Inhibition of inflammatory lymphangiogenesis by integrin alpha5 blockade.

The interaction between endothelial cells and extracellular matrix proteins plays an important role in (hem)angiogenesis. Integrins are able to mediate the outgrowth of newly formed blood vessels. In contrast, the role of integrins in lymphangiogenesis, ie, the outgrowth of new from pre-existing lymphatic vessels, has so far been unclear. Here, expression and functional relevance of integrins on lymphatic endothelium in vivo was investigated using the mouse model of combined inflammatory corneal hemangiogenesis and lymphangiogenesis. Immunohistochemistry revealed novel expression of both integrin alpha5 and alphav on both resting and activated lymphatic vessels in vivo. Integrin alpha5-inhibiting small molecules significantly blocked the outgrowth of new lymphatic vessels into the cornea in a dose-dependent manner. The outgrowth of blood vessels was less significantly affected by this treatment, thus allowing for selective inhibition of lymphangiogenesis at lower dosages. Combined inhibition of integrin alpha5 and alphav using inhibiting molecules did not significantly increase the anti-lymphangiogenic effect in vivo, thus suggesting an important functional role of integrin alpha5 in lymphangiogenesis. In summary, our findings demonstrate novel expression of specific integrins on growing lymphatic endothelial cells in vivo and reveal their functional role during lymphangiogenesis. This opens new treatment options for selective inhibition of lymphangiogenesis, eg, in oncology and transplant immunology.

Bevacizumab as a potent inhibitor of inflammatory corneal angiogenesis and lymphangiogenesis.

PURPOSE: To analyze whether bevacizumab can inhibit inflammatory angiogenesis and lymphangiogenesis in the cornea. Bevacizumab (Avastin; Roche, Welwyn Garden City, UK) is a recombinant, humanized, monoclonal antibody against VEGF-A that has been approved by the U.S. Food and Drug Administration for the treatment of colon carcinomas. METHODS: The mouse model of suture-induced corneal neovascularization was used to assess the antihemangiogenic and antilymphangiogenic effect of bevacizumab by systemic and topical application. Corneal flatmounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and CD31 as a pan-endothelial marker, and blood and lymph vascularized areas were analyzed morphometrically. The inhibitory effect of bevacizumab on lymphatic endothelial cells (LECs) was analyzed with a colorimetric (BrdU) proliferation ELISA. The binding ability of bevacizumab to murine VEGF-A was analyzed by Western blot, ELISA, and surface plasmon resonance. RESULTS: The systemic and topical applications of bevacizumab significantly inhibited the outgrowth of blood (P < 0.006 and P < 0.0001, respectively) and lymphatic (P < 0.002 and P < 0.0001, respectively) vessels. Inhibition of the proliferation of LECs was also significant (P < 0.0001). Western blot analysis, ELISA, and the surface plasmon resonance assay showed that bevacizumab binds murine VEGF-A. CONCLUSIONS: Topical or systemic application of bevacizumab inhibits both inflammation-induced angiogenesis and lymphangiogenesis in the cornea. This finding suggests an important role of VEGF-A in corneal lymphangiogenesis. Bevacizumab may be useful in preventing immune rejections after penetrating keratoplasty or tumor metastasis via lymphatic vessels.

[Cataract and keratoplasty--simultaneous or sequential surgery?]. / Katarakt und Keratoplastik--simultane oder sequenzielle Operation?

BACKGROUND AND PURPOSE: Since the introduction of the triple procedure (simultaneous penetrating keratoplasty [PK], extracapsular cataract extraction [CE] and implantation of a posterior chamber intraocular lens [PCIOL]) in the mid-seventies, there is an ongoing discussion among corneal surgeons concerning the best approach for combined corneal disease and cataract. METHODS: Besides the classical triple procedure (1), two alternative microsurgical approaches are feasible: (2) CE + PCIOL prior to PK and (3) CE + PCIOL after PK. For the refractive results after TRIPLE some intraoperative details are crucial: Trephination of recipient and donor from the epithelial side without major oversize (Guided Trephine System or Nonmechanical Excimer Laser Trephination) should preserve the preoperative corneal curvature. Graft and the PCIOL placed in the bag after continuous curvilinear capsulorhexis should be centered along the optical axis. If possible, performing the capsulorhexis under controlled intraocular pressure conditions prior to trephination may help to minimise the risk of capsular ruptures. RESULTS: The major advantage of the TRIPLE is the faster visual rehabilitation and less efforts for the mostly elderly patients. However, two intraocular interventions with approach (2) and (3) bear an increased risk of infection and suprachoroidal haemorrhage. Approach (2) requires a cornea that is still transparent enough to perform cataract surgery, and the risk of intraocular pressure rise after PK seems to be increased. Approach (3) has the potential of a simultaneous reduction of astigmatism during CE (appropriate location of the incision, simultaneous refractive keratotomies or implantation of a toric PCIOL). Disadvantages may include the loss of graft endothelial cells and the theoretically increased risk of immunological allograft reactions. After TRIPLE, major deviations from target refraction have been reported. However, individual multiple regression analysis may help to minimise this problem with appropriate methods of trephination. Since suture removal after PK may result in major individual changes of the corneal curvature, IOL power calculation for approach (3) requires all sutures to be removed at the time of CE. However, even after complete suture removal the abnormal proportions between anterior and posterior curvatures and/or the irregular topographies after PK may be responsible for marked IOL power miscalculations in the individual case. CONCLUSIONS: The postulated better refractive outcome and better uncorrected visual acuity after the sequential approach is opposed by a markedly delayed visual rehabilitation. For this reason, we consider the TRIPLE procedure including CE via open sky in general anesthesia as the method of choice for combined lens and corneal opacities. Because of the often rapidly progressive nuclear cataracts after PK, we recommend the simultaneous approach in elderly patients with Fuchs' dystrophy even with incipient lens opacities.

Penetrating keratoplasty for endothelial decompensation in eyes with buphthalmos.

PURPOSE: To evaluate the prognosis and complications of penetrating keratoplasty (PKP) for corneal decompensation in eyes with buphthalmos and to analyze the risk factors for graft failure. PATIENTS AND METHODS: Clinical records of 13 adult and three pediatric patients who underwent PKP for endothelial decompensation with a previous diagnosis of congenital glaucoma of a total of 3,663 corneal transplantations performed in our department between January 1987 and December 2001 were reviewed retrospectively. During the study period, a total of 33 PKPs was performed in 20 eyes with buphthalmos. The median age of the patients at the time of PKP was 39 years (range, 3 to 72). All patients had a history of intraocular surgery, including multiple glaucoma surgeries, cataract extraction, and PKP. The impact of pre-, intra-, and postoperative factors on graft failure and duration of graft clarity was analyzed. RESULTS: Fifty-five percent (11/20) of the eyes received only one graft, 25% (5/20) received two, and 20% (4/20) received three grafts. During a mean follow-up of 87.2 months (range, 4.5-72), graft failure occurred in 18 of 33 grafts (54%). Seven (7/18, 39%) had immunologic graft rejection, and 11 (11/18, 61%) had nonimmunologic graft failure. At the end of the follow-up, 75% (15/20) of the eyes had clear grafts. Duration of graft clarity was found to be significantly shorter in regrafts compared with that of primary grafts (27.0 +/- 27.7 versus 56.4 +/- 41.0 months, p= 0.02). After PKP, intraocular pressure (IOP) was uncontrolled in 12 (12/33, 36%) grafts. Nine of 20 eyes (45%) required an average of 3.2 cyclodestructive procedures per eye for pharmacologically resistant elevated IOP. The final postoperative vision improved in 70% (14/20) of the eyes and the best visual acuity postoperatively (75% > or =20/400) was significantly better than the preoperative visual acuity (25% > or =20/400, p= 0.0001). CONCLUSIONS: Endothelial decompensation due to congenital glaucoma is a very rare indication for PKP. The incidence of graft failure is high, and nonimmunologic reasons are the leading causes of graft failure in this high-risk population. Visual acuity can be significantly improved but is usually still very limited by advanced glaucomatous optic nerve damage and amblyopia. Efficient control of IOP before and after PKP is mandatory in eyes with buphthalmos to avoid graft failure and progress of glaucomatous optic nerve atrophy.