Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: Direct correlation with clinical retinopathy of prematurity

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases.

Angiogenesis underlies the majority of eye diseases that result in catastrophic loss of vision. Recent evidence has implicated the integrins alpha v beta 3 and alpha v beta 5 in the angiogenic process. We examined the expression of alpha v beta 3 and alpha v beta 5 in neovascular ocular tissue from patients with subretinal neovascularization from age-related macular degeneration or the presumed ocular histoplasmosis syndrome or retinal neovascularization from proliferative diabetic retinopathy (PDR). Only alpha v beta 3 was observed on blood vessels in ocular tissues with active neovascularization from patients with age-related macular degeneration or presumed ocular histoplasmosis, whereas both alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR. Since we observed both integrins on vascular cells from tissues of patients with retinal neovascularization from PDR, we examined the effects of a systemically administered cyclic peptide antagonist of alpha v beta 3 and alpha v beta 5 on retinal angiogenesis in a murine model. This antagonist specifically blocked new blood vessel formation with no effect on established vessels. These results not only reinforce the concept that retinal and subretinal neovascular diseases are distinct pathological processes, but that antagonists of alpha v beta 3 and/or alpha v beta 5 may be effective in treating individuals with blinding eye disease associated with angiogenesis.

Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Roles of nitric oxide in tumor growth.

A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type parental cells. This growth retardation was reversed by the addition of N-iminoethyl-L-ornithine. In nude mice, however, the tumors from these cells grew faster than those derived from wild-type cells and were markedly more vascularized, suggesting that NO may act as part of a signaling cascade for neovascularization. Recent observations that the generation of NO in human breast and gynecological cancers correlates positively with tumor grade are consistent with this hypothesis. We suggest that NO may have a dual pro- and antitumor action, depending on the local concentration of the molecule.