vasoactive-intestinal-peptide and Corneal-Neovascularization

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.. 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.. 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.. 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.

Topics: alpha-MSH; Animals; Aqueous Humor; Cell Proliferation; Cells, Cultured; Corneal Neovascularization; Disease Models, Animal; Endothelium, Lymphatic; Female; Flow Cytometry; Glycoproteins; Humans; Immunohistochemistry; Lymphangiogenesis; Membrane Transport Proteins; Mice; Mice, Inbred BALB C; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide that downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis. Growth factors also play a role in corneal healing and restoration of tissue homeostasis after wounding. However, whether VIP treatment modulates growth factors to promote healing in the infected cornea remains untested and is the purpose of this study.. C57BL/6 (B6) mice were injected with VIP and mRNA and protein levels, and immunostaining for EGF, FGF, HGF, and VEGF-A were done. Exogenous treatment with a mixture of the growth factors also was tested and levels of cytokines, defensins, and bacterial counts were determined.. Real-time RT-PCR, immunostaining, and ELISA data demonstrated that treatment with VIP enhanced levels of EGF, FGF, and HGF during disease, and that VEGF-A, and associated angiogenic molecules also were increased by VIP. Moreover, immunohistochemical studies confirmed that both epithelial and stromal cells participated in growth factor production. Most notably, treatment with a mixture of EGF, FGF, and HGF after disease onset, prevented corneal perforation when compared with controls. This outcome was associated with downregulation of proinflammatory cytokines such as macrophage inflammatory protein-2 (MIP-2), upregulation of anti-inflammatory cytokines such as TGF-β, and antimicrobials β-defensins 2 and 3, as well as decreased plate counts at 1 day postinfection (p.i.) (P = 0.0001).. Collectively, the data provide evidence that VIP treatment modulates growth factors, angiogenic molecules, and defensins in the infected cornea and that this in turn promotes healing and restoration of tissue homeostasis.

Topics: Animals; Colony Count, Microbial; Cornea; Corneal Neovascularization; Corneal Perforation; Corneal Ulcer; Cytokines; Defensins; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Gene Expression; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vascular Endothelial Growth Factor A; Vasoactive Intestinal Peptide