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.

In squamous cell carcinoma, the levels of nitric oxide (NO) derived from inducible NO synthase (iNOS) and prostaglandin E ₂ (PGE ₂ ) derived from cyclooxygenase‐2 (COX‐2) originated from tumor cells or tumor‐associated inflammatory cells have been reported to correlate with tumor growth, metastasis, and angiogenesis. The present study examined the role of the iNOS signaling pathway in PGE ₂ ‐mediated tumor invasiveness and proliferation in squamous cell carcinoma, A431, and SCC‐9 cells. Cell invasion and proliferation promoted by PGE ₂ were blocked by iNOS silencing RNA or iNOS/guanylate cyclase (GC) pharmacological inhibition. Consistently, iNOS‐GC pathway inhibitors blocked mitogen‐activated protein kinase‐ERK1/2 phosphorylation, which was required to mediate PGE ₂ functions. In vivo , in A431 cells implanted in nude mice, GC inhibition also decreased the tumor proliferation index and ERK1/2 activation. PGE ₂ effects were confined to the selective stimulation of the EP2 receptor subtype, leading to epidermal growth factor receptor (EGFR) transactivation via protein ki‐nase A (PKA) and c‐Src activation. EP2‐mediated ERK1/2 activation and cell functions were abolished by inhibitors of PKA, c‐Src, and EGFR, as well as by inhibiting iNOS pathway. Silencing of iNOS also impaired EGFR‐induced ERK1/2 phosphorylation. These results indicate that iNOS/GC signaling is a down‐stream player in the control of EP2/EGFR‐mediated tumor cell proliferation and invasion.—Donnini, S., Finetti, F., Solito, R., Terzuoli, E., Sacchetti, A., Morbidelli, L., Patrignani, P., Ziche, M. EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways. FASEB J. 21, 2418–2430 (2007)

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium–NO adducts was documented by electronic absorption, FT-IR spectroscopy and ¹H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-Br-cGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Cerebral amyloid angiopathy (CAA) caused by amyloid β (Aß) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Aß properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Aß peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild‐type (WT) Aß and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo , in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC ₅₀ of 6.1 and 4.7 μM, respectively. The 2.5 μM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. ß‐Galactosidase staining and the cyclindependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro , WT and E22Q reduced endothelial cell survival with an IC ₅₀ of 12.3 and 8.8 μM, respectively. The 5 μM concentration, devoid of acute effects on the endothelium, was applied chronically to long‐term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with ß‐galactosidase accumulation, down‐regulation of telomerase reversetranscriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Aß treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Aß‐induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.—Donnini, S., Solito, R., Cetti, E., Corti, F., Giachetti, A., Carra, S., Beltrame, M., Cotelli, F., Ziche, M. Aß peptides accelerate the senescence of endothelial cells in vitro and in vivo , impairing angiogenesis. FASEBJ. 24, 2385–2395 (2010). www.fasebj.org