Semaphorin 3F Modulates Corneal Lymphangiogenesis and Promotes Corneal Graft Survival.

Purpose: Corneal vascularization significantly increases the risk for graft rejection after keratoplasty. Semaphorin 3F (Sema3F) is a known modulator of physiologic avascularity in the outer retina. The aim of this study was to investigate whether Sema3F is involved in maintaining corneal avascularity and can reduce the risk for corneal graft rejection. Methods: Corneal Sema3F expression was investigated using immunohistochemistry and qPCR in human and murine tissue. Pathologic invasion of blood and lymph vessels into corneal tissue was analyzed in the murine corneal suture and high-risk keratoplasty model. The anti-lymphangiogenic effects of Sema3F were further investigated using an in vitro spheroidal sprouting model with supernatant from isolated primary human corneal epithelial cells (hCECs). Results: Sema3F is constitutively expressed in human and murine corneal epithelium. In the corneal suture model, lymphangiogenesis was significantly suppressed by topical Sema3F treatment (P = 0.0003). In the murine high-risk keratoplasty model, pretreatment by topical Sema3F in the inflammation phase significantly promoted subsequent graft survival (P = 0.0006). In this model, both lymph- and blood angiogenesis were reduced (P < 0.05). In vitro, hCEC supernatant had a direct anti-lymphangiogenic effect on human lymphatic endothelial cells (P < 0.01). This effect was completely abolished by addition of anti-Sema3F antibodies. Conclusions: Sema3F is a novel mediator of corneal avascularity with potent anti-lymphangiogenic properties. Topical treatment with Sema3F eye drops may help to limit corneal vascularization and improve outcomes in high-risk keratoplasty patients.

Fine Needle-Diathermy Regresses Pathological Corneal (Lymph)Angiogenesis and Promotes High-Risk Corneal Transplant Survival.

Pathological corneal hem- and lymphangiogenesis are prime risk factors for corneal graft rejection. Fine needle-diathermy (FND) is an option to regress corneal blood vessels; however, whether this treatment besides clinically visible blood vessels also affects invisible lymphatic vessels is so far unknown. Here we test the hypothesis that FND destroys not only blood but also lymphatic vessels, thereby promotes corneal high-risk graft survival. The effect of FND was studied in vivo using BALB/c mice and the model of suture-induced corneal neovascularization. Mice were divided into three groups: FND, ANTI (anti-inflammatory therapy) and NON (control). Five, 7, 10 and 20 days after cauterization, corneas were harvested and stained with LYVE-1, CD31 to quantify (lymph)angiogenesis. The long-term survival of allografts was compared between the three groups. FND caused significant regression of both blood and lymphatic vessels compared to the control group at all time points (p < 0.05) with the most obvious effect at day 7 (p < 0.01). Graft survival was significantly prolonged when transplants were placed into the FND pretreated group (p < 0.0001). The effect of the anti-inflammatory therapy alone was less effective compared to FND (p < 0.05). This novel lymphangioregressive effect of FND can be used clinically to precondition high-risk recipients to promote graft survival.

Photodynamic Therapy Leads to Time-Dependent Regression of Pathologic Corneal (Lymph) Angiogenesis and Promotes High-Risk Corneal Allograft Survival.

Purpose: Pathologic corneal (lymph) angiogenesis is a known risk factor for immune-mediated allograft rejections after corneal transplantation. However, there is no established treatment to regress pre-existing pathological corneal blood and lymphatic vessels. This study assessed the possibility to regress both vessel types by photodynamic therapy (PDT) after intravenous (i.v.) verteporfin injection, the influence of timing of PDT after verteporfin injection, and the effect on graft survival in high-risk keratoplasty. Methods: BALB/c mice were used for suture-induced inflammatory corneal neovascularization to induce combined hem- and lymphangiogenesis. The treated group received PDT 3 minutes, 1 hour, and 24 hours after an i.v. verteporfin injection (control group: phosphate buffered saline). Corneal flatmounts were excised 3 days, 1 week, and 2 weeks after corneal PDT and stained with cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 antibodies (LYVE-1) to quantify hem- and lymphangiogenesis. Graft survival rates were compared between high-risk recipients with and without preoperative PDT. Results: Corneal blood vessels were significantly reduced when PDT was performed 3 minutes after i.v. verteporfin injection, whereas lymphatic vessels showed no significant difference. Both blood and lymphatic vessels were regressed when PDT was performed 1 hour or 24 hours after i.v. verteporfin application. Long-term allograft survival increased significantly in PDT-pretreated eyes when compared with controls. Conclusions: PDT after i.v. verteporfin injection can selectively regress pre-existing corneal blood vessels or both blood and lymphatic vessels depending on the timing of PDT after verteporfin injection. The pretreatment of recipients with PDT and i.v. verteporfin might be a promising new method to improve graft survival in high-risk eyes.

Identification of Novel Endogenous Anti(lymph)angiogenic Factors in the Aqueous Humor.

Purpose: The avascular cornea is in direct contact with aqueous humor (AqH). Here we investigate whether AqH exerts anti(lymph)angiogenic effects and thereby may contribute to corneal (lymph)angiogenic privilege. Methods: Using the murine model of suture-induced inflammatory corneal hem- and lymphangiogenesis, the potential anti(lymph)angiogenic effect of AqH was analyzed by applying murine AqH as eyedrops. Anti(lymph)angiogenic effects were measured using morphometric analysis of flat mounts stained with CD31 as panendothelial and LYVE-1 as specific lymphatic endothelial marker. The potential antilymphangiogenic effect of immunomodulatory factors contained in AqH such as vasoactive intestinal peptide (VIP) and α-melanocyte stimulating hormone (α-MSH) was analyzed in lymphatic and blood vascular endothelial cell proliferation assays in vitro. Results: Topically applied AqH significantly inhibited corneal hemangiogenesis and even more so lymphangiogenesis in vivo and directly in vitro. The immunoregulatory factors VIP and α-MSH significantly inhibited lymphatic endothelial cell proliferation in vitro. Depletion of VIP or α-MSH from AqH diminished its anti-hem- and lymphangiogenic potential. Conclusions: Aqueous humor exerts significant antilymphangiogenic effects in vivo. This is at least partially mediated by the known immunomodulatory factors VIP and α-MSH present in the AqH. Therefore, AqH not only contributes to corneal lymphangiogenic privilege and is a new tool to identify novel endogenous regulators of lymphangiogenesis but also may have therapeutic applications.

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8+ T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.