Angiogenic Molecules

Vascular endothelial growth factor

VEGF is a dimeric 46-kDa glycoprotein. This growth factor stimulates angiogenesis by increasing EC proliferation, migration, proteolytic activity, and capillary tube formation. It also significantly increases vascular permeability. The VEGF family includes VEGF-A, - B, - C, - D, placenta growth factor (PlGF), and the viral VEGF homolog VEGF-E. VEGF-B promotes nonangiogenic tumor progression, while VEGF-C and - D participate in angiogenesis and lymphangiogenesis. VEGF-A also participates in angiogenesis and increases vascular permeability.

Five isoforms of VEGF-A (VEGF115,121, 165,189, and 206) can be generated by alternative splicing of the same gene. The longer isoforms (VEGF189 and 206) are matrix-bound, whereas the shorter isoforms (VEGF121 and 165) are freely diffusible. These VEGF isoforms produce different actions when secreted. For example, all isoforms increase in vascular permeability, but only VEGF121 and VEGF165 have mitogenic activity. VEGF121 has greater angiogenic activity than VEGF165 or VEGF189. On the other hand, VEGF165 is more potent than VEGF121 in induction of inflammation, intercellular adhesion molecule-1 (ICAM-1) expression in ECs, and chemotaxis of monocytes. This suggests that alternate splicing of VEGF messenger RNA (mRNA) can be regulated to achieve a range ofphysiologic actions.

The VEGF family members act through binding to high-affinity receptor tyrosine kinases. Two high-affinity receptor tyrosine kinases have been identified for VEGF-A: VEGFR-1 (fms-like tyrosine kinase-1 or Flt-1) and VEGFR-2 (kinase insert domain-containing receptor or KDR). VEGFR-3 (fms-like tyrosine kinase-4 or Flt-4) serves as a high-affinity receptor for VEGF-C and - D. Both VEGFR-1 and -2 are expressed primarily in vascular ECs, while VEGFR-3 is predominantly expressed in lymphatic ECs. VEGF-B binds to VEGFR-1 and has mild mitogenic activity. In contrast, binding of VEGF-D and - C to VEGFR-3 regulates the growth and differentiation of blood vessels and lymphatic endothelium.

VEGF is produced by macrophages, T cells, astrocytes, pericytes, fibroblasts, retinal pigment epithelial cells, and smooth muscle cells. In addition, VEGF is expressed in all three cellular layers of the cornea. It is highly expressed in vascular ECs of limbal vessels and in new stromal vessels. Under inflammatory conditions, VEGF expression is increased in epithelial and vascular ECs, particularly near macrophage infiltrates and fibroblasts in corneal scars. Following corneal cautery, VEGF165 and 189 mRNA is increased at 48 h and returns to baseline by day 7. Immunohistochemistry has revealed that VEGF is initially expressed in neutrophils and later expressed in macrophages, demonstrating that VEGF production by leukocytes is associated with corneal NV. In addition, VEGF concentration is significantly increased in vascularized corneas as compared to normal corneas. In limbal-deficiency-induced corneal NV, VEGF mRNA and protein are induced after injury and are both temporally and spatially correlated with inflammation and NV. VEGF is not only induced during NV, but is also required for corneal angiogenesis. The indispensable role of VEGF in angiogenesis is shown by the finding that stromal implantation of anti-VEGF antibodies inhibits NV in a rat model. Conversely, implantation of a Hydron pellet containing VEGF into the stroma induces severe corneal NV without significant inflammation.

The effects ofVEGF in the cornea are not limited to NV, as this growth factor has also been shown to regulate goblet cell migration. Studies analyzing the correlation between cornea NV and conjunctivalization showed that VEGFR-1 is present in the conjunctiva-like epithelium covering the cornea as well as in goblet cells, invading leukocytes, and the corneal vasculature. Inhibition of VEGF activity inhibited not only corneal NV, but also goblet cell density, suggesting that VEGF may promote goblet cell migration.

Evidence suggests that VEGF also participates in corneal lymphangiogenesis. Corneal lymphangiogenesis may contribute to graft sensitization and rejection, following high-risk keratoplasty of vascularized corneas. VEGF-C binds to VEFGR-3 and induces lymphatic growth in the cornea. Interestingly, inhibition of lymphatic growth is observed after administration of a VEGF trap that neutralizes VEGF-A, but not VEGF-C or - D. This could be explained by the chemotactic effect on macrophages that release VEGF-C in inflamed corneas observed with VEGF-A. Thus, VEGF-A amplifies signals essential for lymphatic growth. In general, corneal lymphangiogenesis seems to correlate well with the degree of corneal hemangiogenesis.

Recent studies have shown that VEGF, although present in the cornea, does not promote angiogenesis under normal conditions. VEGF-A found in corneal tissue is mostly bound to an alternative spliced secreted isoform of VEGFR-1 (sflt-1), which acts as a trap for secreted VEGF-A and in this way contributes to maintenance of corneal avascularity. In addition, VEGFR-3 is expressed in endothelial as well as epithelial cells in the cornea. When VEGF-C and - D bind to endothelial VEGFR-3, they stimulate proangiogenic signaling. In contrast, VEGFR-3 expressed by corneal epithelium acts as a decoy receptor sequestering VEGF but yet rendering it available when an angiogenic response is needed to enhance the immune defense. This VEGFR-3 sink system is a potent mechanism that inhibits inflammatory-induced angiogenesis.

Basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is another potent angiogenic factor. It is a member of the fibroblast growth factor (FGF) family, which includes 23 heparin-binding peptides widely expressed during cell differentiation, angiogenesis, mitogenesis, and wound healing. bFGF functions are mediated by the receptors FGFR-1, -2, -3, and-4. FGF recptor-1 (FGFR-1) is expressed in normal corneal epithelium, while bFGF is upregulated following injury. It is also upregulated following co-culture of corneal epithelial cells with vascular EC and keratocytes. The affinity of bFGF for its receptor differs according to the extent of maturation of new vessels. This may be due to varying expression of heparan sulfate proteoglycans and highlights the role of ECM proteins in the regulation of corneal angiogenesis.

Matrix metalloproteinases

The matrix metalloproteinases (MMPs) constitute a multigene family of zinc-binding proteolytic enzymes that participate in ECM remodeling. Many of the growth factors that modulate angiogenesis also influence MMP expression. These growth factors include VEGF, FGF-2, and tumor necrosis factor-alpha (TNF-a). Vascular ECs respond by secreting proteolytic enzymes that degrade the ECM to facilitate migration and differentiation ofECs. The MMPs that have identified in the cornea are collagenases I and II (MMP-1 and -13), stromelysin (MMP-3), matrily-sin (MMP-7), membrane-type MMP (MT-MMP-14), and gelatinases A and B (MMP-2 and -9). Both MMP-2 and MMP-9 are proteolytically activated primarily by MT1-MMP during capillary formation. Several reports suggest that these MMPs participate in vascular invasion by directly degrading the matrix or releasing matrix-bound cytokines and growth factors. Accordingly, inhibition of MMP-9 activity in the cornea decreases angiogenesis. However, given their ability to degrade ECM, MMPs exhibit a dual action in angiogenesis. For example, MMP-2 activation may release anti-angiogenic fragments, allow the production of potent angiostatic factors, or facilitate angiogenesis.

Lipid mediators

One ofthe initial events that occurs after corneal injury is the release of arachidonic acid. In the corneal epithelium, ara-chidonic acid is then metabolized by cyclooxygenase (COX) to generate eicosanoids (such as 12- and 15-HETE), lipoxin A4 (LXA4), and prostaglandins. 12(S)-HETE is a powerful angiogenic factor, and COX inhibitors have been shown to reduce corneal angiogenesis in animal models. Plateletactivating factor is another potent lipid mediator released from the cell membrane after corneal injury. It contributes to corneal NV by increasing expression of VEGF, MMP-9, and urokinase plasminogen activator (uPA), all of which subsequently stimulate vascular EC migration.

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