Experimental & Molecular Medicine is an open access journal that publishes the highest quality articles in translational research and biomedical studies.

Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.

In order to direct antisense oligonucleotides to specific tissues or cell types in vivo, we are exploring the possibility to utilize lipoproteins as transport vehicles. A 16-mer oligonucleotide (ODN) was derivatized at the 5' prime through a ³²P phosphate spacer with cholesterol, yielding a ³²P labeled amphiphatic cholesteryl..oligonucleotlde (cholODN). Incubation of cholODN with low-density lipoprotein (LDL) for 2 hr at 37°C resulted in the formation of a cholODN-LDL complex that migrates as a single peak on agarose gel electrophoresis. The cholODN was found to bind quantitatively to both high-density lipoproteins (HDL) and LDL, but not to albumin. Stable oligonucleotide LDL particles with up to 50 molecules of cholODN per LDL particle coutd be obtained. In contrast, the control ODN did not show affinity for plasma lipoproteins. Upon injection into rats, cholODN became rapidly associated with plasma lipoproteins while control ODNs were recovered in the lipoprotein deficient serum fraction. The plasma half-life of cholODN (9–11 min) Is considerably prolonged as compared with the control ODN (t1/2 <1 min). The cholODN-LDL was at least 5 min stable against degradation by rat plasma nucleases. It is concluded that derivatization of antisense oligonucleotides with cholesterol profoundly modifies their in vivo fate and opens possibilities for efficient and specific receptor-dependent targeting, mediated by lipoproteins coupled with specific recognition markers to various hepatic cell types.

Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.