interleukin-8 and Corneal-Neovascularization

Previously, we have reported that the Secreted Ly6/uPAR related protein-1 (SLURP1) serves an important immunomodulatory function in the ocular surface. Here, we examine the involvement of SLURP1 in regulating corneal angiogenic privilege. Slurp1 expression detected by QPCR, immunoblots and immunofluorescent stain, was significantly decreased in mouse corneas subjected to alkali burn-induced corneal neovascularization (CNV). Addition of exogenous SLURP1 (6XHis-tagged, E. coli expressed and partially purified using Ni-ion columns) significantly suppressed the tumor necrosis factor-α (TNF-α)-stimulated human umbilical cord vascular endothelial cell (HUVEC) tube formation. SLURP1 suppressed the HUVEC tube length, tube area and number of branch points, without affecting their viability and/or proliferation. Exogenous SLURP1 in HUVEC also suppressed the TNF-α-induced (i) interleukin-8 (IL-8) and TNF-α production, (ii) adhesion to different components of the extracellular matrix, (iii) migration, and (iv) nuclear localization of NFκB. Together, these results demonstrate that SLURP1 suppresses HUVEC tube formation by blocking nuclear translocation of NFκB, and suggest a potential role for SLURP1 in promoting corneal angiogenic privilege.

Topics: Angiogenesis Inhibitors; Animals; Antigens, Ly; Burns, Chemical; Cell Adhesion; Cell Movement; Cell Proliferation; Corneal Injuries; Corneal Neovascularization; Disease Models, Animal; Eye Burns; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Mice; NF-kappa B; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Topics: Animals; Arsenicals; Blister; Blood Vessels; Chemical Warfare Agents; Cornea; Corneal Keratocytes; Corneal Neovascularization; Corneal Pachymetry; Corneal Stroma; Cyclooxygenase 2; Epithelium, Corneal; Interleukin-8; Keratitis; Matrix Metalloproteinase 9; Rabbits; Risk Assessment; Time Factors; Vascular Endothelial Growth Factor A

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.

Topics: Animals; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Corneal Neovascularization; Endothelial Cells; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Neovascularization, Physiologic; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Transfection; Up-Regulation

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

Topics: Alleles; Amino Acid Sequence; Angiostatic Proteins; Animals; Blood Platelets; Cells, Cultured; Chemotaxis; Corneal Neovascularization; Culture Media, Conditioned; Cytoplasmic Granules; Endothelial Cells; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Humans; Interleukin-8; Molecular Sequence Data; Platelet Activation; Platelet Factor 4; Rats; Structure-Activity Relationship; Thrombin

In diabetes mellitus, there is a problem of both premature atherosclerosis as well as impaired collateralization. Studies were performed using the rat corneal angiogenesis model as a surrogate for collateralization to determine the effect of diabetes mellitus on endothelin (ET)-1, ET-3, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8)-mediated angiogenesis. In an initial group of experiments, streptozotocin-induced diabetes resulted in impairment of ET-1-mediated angiogenesis from 69% to 32%, but was only impaired from 74% to 59% for ET-3. When rats were fluid-resuscitated, mortality fell, and the incidence of inhibition of angiogenesis decreased for ET-1, but was still at 47%. Inhibition of ET-3-mediated angiogenesis in fluid-resuscitated rats was essentially unaffected from 74% to 75%. Studies of VEGF and IL-8 in fluid-resuscitated rats demonstrated that VEGF-mediated angiogenesis was only inhibited from 49% to 45%, but there was inhibition of IL-8-mediated angiogenesis from 62% to 31%. We concluded that there may be two mechanisms by which ET-1-mediated corneal angiogenesis is inhibited: a decrease in intravascular volume and dynamic forces affecting angiogenesis, and a direct effect of diabetes on some aspect of cell growth or angiogenic process. Diabetes also appeared to inhibit IL-8-mediated angiogenesis, but had very little or no effect on ET-3- or VEGF-mediated angiogenesis.

Topics: Animals; Corneal Neovascularization; Diabetes Mellitus, Experimental; Endothelial Growth Factors; Endothelin-1; Endothelin-3; Endothelins; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lymphokines; Models, Animal; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Division; Chemokines, CXC; Chemotaxis; Corneal Neovascularization; Endothelium, Vascular; Humans; Interferon-gamma; Interleukin-8; Lung Neoplasms; Microcirculation; Mutagenesis, Site-Directed; Neoplasms; Neovascularization, Pathologic; Rats; Recombinant Proteins