Systemic administration of the nitric oxide (NO) synthase inhibitor Nomega-nitro--arginine methyl ester (L-NAME) to rabbits bearing a corneal implant blocked vascular endothelial growth factor (VEGF), but not basic fibroblast growth factor (bFGF)-induced angiogenesis. L-NAME completely blocked angiogenesis induced by VEGF-transfected MCF-7 breast carcinoma cells and the cells remained dormant in the cornea. Postcapillary endothelial cell migration and growth induced by VEGF were blocked by both the NO synthase inhibitor Nomega-mono-methyl--arginine and by the guanylate cyclase inhibitor LY 83583. We conclude that NO is a downstream imperative of VEGF-, but not bFGF-induced angiogenesis, and propose that the NO synthase/guanylate cyclase pathway is a potential target for controlling tumor angiogenesis in response to VEGF. Our studies support recent evidence that VEGF and bFGF induce angiogenesis by different mechanistic pathways using the alphavbeta5 and alphavbeta3 integrins, respectively.

Angiogenesis, the development of new capillaries form pre-existing vessels, requires the coordinate activation of endothelial cells, which migrate and proliferate in response to growth factors to form functional vessels. Therapeutic angiogenesis is proposed to restore tissue integrity and function following damage and ischemia, while strategies aimed to block or suppress the neovascular growth are designed as adjuvant therapies for cancer treatment. Different experimental and clinical observations support the existence of a molecular / biochemical link between vasodilation, nitric oxide (NO) production and angiogenesis. NO significantly contributes to the prosurvival / proangiogenic program of capillary endothelium by triggering cell growth and differentiation via endothelial-constitutive NO synthase (ecNOS) activation, and cyclic GMP (cGMP) dependent gene transcription. Re-establishment of a balanced NO production in the cardiovascular system results in a reduction of cell damage during inflammatory and vascular diseases. Elevation of NOS activity in correlation with angiogenesis and tumor growth and aggressiveness has been extensively reported in experimental and human tumors. On these bases, the nitric oxide pathway appears to be a promising target for the development of pro- and anti-angiogenic therapeutic strategies. In particular, the use of NOS inhibitors or NO scavengers seems appropriate to reduce edema, block angiogenesis and facilitate antitumor drug delivery.

The hypothesis that thrombospondin‐1 (TSP‐1) can exert opposite effects on angiogenesis depending on the functional status of its domains/fragments was investigated. In the rabbit cornea, TSP‐1 inhibited angiogenesis induced by fibroblast growth factor‐2 (FGF‐2). However, when tested per se, TSP‐1 was able to elicit an angiogenic response comparable to that induced by FGF‐2. Induction of angiogenesis was dose‐dependent (20 ng ‐ 2 μg/pellet), was prevented by anti‐TSP antibodies or by heat‐inactivation of TSP‐1, and was not due to inflammatory mediators, to FGF‐2 or to TGF‐β. Equimolar concentrations of the 25 kDa heparin binding fragment of TSP‐1 were even more efficient than the whole molecule, and promoted the angiogenic activity of FGF‐2. On the contrary, the 140 kDa fragment of TSP‐1 did not induce angiogenesis and turned off the angiogenic response to FGF‐2. The 25 kDa fragment and TSP‐1, but not the 140 kDa fragment, increased endothelial cell invasiveness and stimulated the production and activation of matrix metalloproteinase‐2 (MMP‐2). Moreover, the 25 kDa fragment reduced the synthesis of the MMP‐2 inhibitor TIMP‐2, while the 140 kDa fragment caused a twofold increase in TIMP‐2 production and inhibited MMPs stimulation by TSP‐1 and FGF‐2. We conclude that TSP‐1 is a source of smaller mediators of angiogenesis, which affect in an opposite way endothelial cell functions and proteolytic activity, thus resulting in an opposite final effect on angiogenesis.

Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.

Reactive oxygen species (ROS) are produced as a result of aerobic metabolism and as by‐products through numerous physiological and biochemical processes. While ROS‐dependent modifications are fundamental in transducing intracellular signals controlling pleiotropic functions, imbalanced ROS can cause oxidative damage, eventually leading to many chronic diseases. Moreover, increased ROS and reduced nitric oxide (NO) bioavailability are main key factors in dysfunctions underlying aging, frailty, hypertension, and atherosclerosis. Extensive investigation aims to elucidate the beneficial effects of ROS and NO, providing novel insights into the current medical treatment of oxidative stress‐related diseases of high epidemiological impact. This review focuses on emerging topics encompassing the functional involvement of aquaporin channel proteins (AQPs) and membrane transport systems, also allowing permeation of NO and hydrogen peroxide, a major ROS, in oxidative stress physiology and pathophysiology. The most recent advances regarding the modulation exerted by food phytocompounds with antioxidant action on AQPs are also reviewed.

Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.