Under normal physiological conditions, angiogenesis (blood vessel formation) and lymphangiogenesis (lymphatic vessel formation) are tightly regulated processes that occur exclusively during embryonic development, pregnancy, and wound healing. Vascular endothelial growth factor (VEGF) and VEGF receptors are key mediators of this regulation. VEGF-A,-B,-C, and -D, and VEGF receptors 1, 2, and 3 differ in both their expression profiles and their functions. In addition, multiple variants of VEGF ligands and VEGF receptors exist that are produced by alternative splicing or cleavage. VEGF R1 (also called Flt-1) and VEGF R2 (also called Flk-1 or KDR) are angiogenic type I transmembrane receptors that primarily respond to VEGF-A.1, 2, 3 Although the affinity of VEGF-A is highest for VEGF R1, VEGF R2 appears to be the primary mediator of VEGF angiogenic activity.1
||Molecular Mechanisms Regulating Vessel Growth in the Cornea. A. Angiogenesis and lymphangiogenesis are regulated by different forms of VEGF binding to three receptor tyrosine kinases, VEGF R1, VEGF R2, and VEGF R3. VEGF-A binding to VEGF R2 primarily regulates angiogenesis, while VEGF-C binding to VEGF R3 regulates lymphangiogenesis. These processes can be inhibited by soluble forms of the VEGF receptors, and by anti-VEGF or anti-VEGF R antibodies. B. The role of a 75 kDa form of sVEGF R2 in lymphangiogenesis has recently been revealed by studying its effects in the cornea of the eye. In the normal cornea, blood and lymphatic vessels are present in the sclera but do not significantly invade the cornea. If sVEGF R2 (75 kDa) is not present during development, the newborn cornea is invaded by lymphatic vessels, but not by blood vessels. Lymphatic vessel invasion can be inhibited by either ectopic expression of sVEGF R2 (75 kDa) or administration of a VEGF-C antibody. These studies suggest that anti-VEGF R2 antibodies may have variable effects on angiogenesis and lymphangiogenesis depending on which forms of the receptor are recognized by the antibody
In contrast, lymphatic development is associated with the response of VEGF R3 to VEGF-C. While the effects of VEGF signaling on angiogenesis have been studied extensively, the mechanisms controlling lymphangiogenesis are much less clear.1, 2, 3
VEGF signaling is regulated by soluble forms of both VEGF R1 (sVEGF R1) and VEGF R2 (sVEGF R2). In 2006, Ambati et al. demonstrated that sVEGF R1 was an anti-angiogenic splice variant that since has been shown to play a role in preeclampsia during pregnancy.3, 4 In contrast, relatively little has been discovered about sVEGF R2. sVEGF R2 was first reported by Ebos et al. in 2004.5 It was identified to be a 160 kDa protein in the circulation, but it was not clear whether this form is a proteolytic cleavage product or an alternatively spliced isoform, or whether it is a monomer, a homodimer, or a heterodimer.1, 2, 5, 6 A recent paper by Albuquerque et al. sheds new light on the regulation of lymphatic vessel growth by characterizing the expression and function of a 75 kDa soluble form of VEGF R2.7 This form has surprisingly low affinity for VEGF-A, but instead binds to VEGF-C and competes with VEGF R3 to inhibit lymphangiogenesis.7
As an avascular tissue, the cornea of the eye has been widely used to study the control of both angiogenesis and lymphangiogenesis.2, 3 In the adult mouse cornea, sVEGF R2 (75 kDa) is found only at the periphery, but it is expressed at a much higher level in newborn mice.7 Tissue-specific loss of corneal sVEGF R2 (75 kDa) expression in newborn mice leads to abundant lymphangiogenesis without corresponding angiogenesis, similar to what is observed if VEGF-C is overexpressed. Treatment of corneal transplants with monomeric sVEGF R2 (75 kDa) blocks only lymphangiogenesis, in contrast to soluble dimeric VEGF R2-Fc, which inhibits both lymphatic and blood vessel formation. Both forms are effective in decreasing the frequency of corneal transplant rejection, possibly due to reduced lymphatic communication with the immune system. Albuquerque et al. identified a similar activity associated with sVEGF R2 (75kDa) in the human cornea.7 The human form was also shown to be effective in blocking VEGF-C-mediated proliferation of human lymphangiomas in vitro.7
Studies performed using exogenous sVEGF R2 or blocking antibodies directed against VEGF R2 have generated conflicting results. 5, 6, 7, 8, 9, 10 Albuquerque et al. provide a possible explanation. They propose that in contrast to the 75 kDa monomeric form of sVEGF R2, Fc-linked recombinant VEGF R2 and possibly other endogenous dimeric soluble forms, retain the ability to bind to VEGF-A and interfere with angiogenesis.7 Blocking antibodies to VEGF R2, depending on their recognition site, may therefore affect the transmembrane form, one or more soluble forms, or multiple forms, producing entirely different results depending on which form is inhibited. VEGF R2 antibodies are being evaluated in clinical trials as anti-angiogenic agents for the treatment of breast, prostate, ovarian, renal, skin, and hepatic cancers. These antibodies appear to inhibit tumor angiogenesis as expected, but may be less effective as anti-lymphangiogenic agents. 3, 9, 10, 11 Since lymphatic vessels allow cancer cell migration to the lymph nodes, it is conceivable that these antibodies could have unintended effects on cancer dissemination.2
- Olsson, A-K. et al. (2006) Nat. Rev. Mol. Cell Biol. 7:359.
- Cueni, L.N. & M. Detmar (2008) Lymphat. Res. Biol. 6:109.
- Lohela, M. et al. (2009) Curr. Opin. Cell Biol. 21:154.
- Ambati, B.K. et al. (2006) Nature 443:993.
- Ebos, J.M.L. et al. (2004) Mol. Cancer Res. 2:315.
- Ebos, J.M.L. et al. (2008) Cancer Res. 68:521.
- Albuquerque, R.J.C. et al. (2009) Nat. Med. 15:1023.
- Goldman, J. et al. (2007) FASEB J. 21:1003.
- Sini, P. et al. (2008) Cancer Res. 68:1581.
- Burton, J.B. et al. (2008) Cancer Res. 68:7828.
- Krupitskaya, Y. & H.A. Wakelee (2009) Curr. Opin. Invest. Drugs 10:597.
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