Raffaella Solito

Prostaglandin E2 Regulates Angiogenesis via Activation of Fibroblast Growth Factor Receptor-1

Prostaglandin E2 (PGE2) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE2 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. PGE2 (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. PGE2 stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Gαi subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE2 did not phosphorylate FGFR-1. Upon binding to its receptor, PGE2 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 PGE2. 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, PGE2 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 PGE2 angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways

In squamous cell carcinoma, the levels of nitric oxide (NO) derived from inducible NO synthase (iNOS) and prostaglandin E2 (PGE2) 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 PGE2‐mediated tumor invasiveness and proliferation in squamous cell carcinoma, A431, and SCC‐9 cells. Cell invasion and proliferation promoted by PGE2 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 PGE2 functions. In vivo, in A431 cells implanted in nude mice, GC inhibition also decreased the tumor proliferation index and ERK1/2 activation. PGE2 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)

Aβ peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis

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 IC50 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 IC50 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

Fibroblast Growth Factor-2 Mediates Angiotensin-Converting Enzyme Inhibitor-Induced Angiogenesis in Coronary Endothelium

The beneficial effect exerted by angiotensin-converting enzyme inhibitors (ACEI) on vascular endothelium has been attributed to restoration of endothelial cell survival properties and improvement of angiogenesis. Fibroblast growth factor (FGF)-2 is an angiogenic factor for the microvascular endothelium, which tonically promotes endothelial cell growth and survival through an autocrine/paracrine mechanism. Here, we formulate the hypothesis that FGF-2 might contribute to the prosurvival/proangiogenic effect of ACEI. We investigated zofenoprilat and, in selected experiments, lisinopril, as representatives of ACEI. These compounds induced formation of pseudocapillaries in vessel fragments isolated from porcine coronary and human umbilical arteries by increasing endothelial cell growth up to 5-fold. Angiogenesis was abolished by inhibitors of nitric-oxide synthase (NOS) pathway and by anti-FGF-2 antibodies. Consistently, in cultured coronary endothelial cells (CVECs), ACEI up-regulated endothelial NOS (eNOS) and FGF-2 and induced mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 activation. The overexpression of eNOS/FGF-2 produced, at the functional level, enhanced cell proliferation and migration, the latter effect being dose-dependent and maximal at 0.1 μM zofenoprilat. The importance of FGF-2 for the acquisition of the angiogenic phenotype elicited by ACEI was clearly demonstrated by the impairment of endothelial functions following transfection of CVECs with small interference RNA for FGF-2. Moreover, FGF-2 silencing greatly affected the nuclear translocation of the FGF receptor (FGFR)-1, highlighting the autocrine mode of action of FGF-2. At the endothelial membrane level, zofenoprilat appeared to activate the bradykinin B1 receptor, a known stimulant of FGF-2 expression. In conclusion, we show that ACEI exert protective/proangiogenic effects in microvascular coronary endothelial cells by activating the endogenous FGF-2/FGFR-1 system.

Dutch and arctic mutant peptides of β amyloid1–40 differentially affect the FGF-2 pathway in brain endothelium

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Abeta on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Abeta structure, suggesting that the toxic effect of the Abeta assemblies goes beyond mere multimerization.

Pyrazolo-pyrimidine-derived c-Src inhibitor reduces angiogenesis and survival of squamous carcinoma cells by suppressing vascular endothelial growth factor production and signaling.

Src tyrosine kinase family cooperates with activated growth factor receptors to regulate growth, invasion and metastasis. The authors examined the influence of a novel c‐Src inhibitor, 1l, derived from 4‐amino‐substituted‐pyrazolo–pyrimidines, on tumor angiogenesis and on the angiogenic output of squamous carcinoma cells, A431 and SCC‐4. The effect of 1l was assessed on growth and microvessel density in A431 tumors and its effect compared with the established c‐Src inhibitor PP‐1. The effects of c‐Src inhibition were investigated on vascular endothelial growth factor (VEGF) expression and activity in tumor cells grown in vivo and in vitro, as well as on VEGF mediated signaling and on endothelial cell functions. Nanomolar concentrations of 1l decreased tumor volume promoted by A431 implanted in nude mice, without affecting in vitro cell tumor survival. This effect was related to 1l inhibition of VEGF production, and secondary to an effect on tumor microvessel density. The rabbit cornea assay confirmed that 1l markedly decreased neovessel growth induced by VEGF. In cultured endothelial cells, 1l inhibited the VEGF‐induced phosphorylation on tyr416 of c‐Src, resulting in a reduced cell proliferation and invasion. Consistently, 1l dowregulated endothelial nitric oxide synthase, MAPK‐extracellular receptor kinase 1–2 (ERK1‐2) activity and matrix metalloproteinases (MMP‐2/MMP‐9), while the tissue inhibitors of metalloproteinases (TIMP2/TIMP‐1) were upregulated. These results demonstrate that nM concentrations of c‐Src kinase inhibitors (1l and PP‐1), by reducing the production of VEGF released by tumor cell and its endothelial cell responses, have a highly selective antiangiogenesis effect, which might be useful in combination therapies.

Mitochondrial aldehyde dehydrogenase-2 activation prevents β-amyloid-induced endothelial cell dysfunction and restores angiogenesis.

Summary Amyloid &bgr; peptides (A&bgr;1–40 and A&bgr;1–42) cause cerebral degeneration by impairing the activity of angiogenic factors and inducing apoptosis and senescence in the endothelium. Amyloid peptides are known to induce oxidative stress. Impairment of mitochondrial aldehyde dehydrogenase 2 (ALDH2) following oxidative stress, results in accumulation of toxic aldehydes, particularly 4-hydroxynoneal (4-HNE). We sought to determine the role of mitochondrial ALDH2 in A&bgr;-related impairment of angiogenesis. We hypothesized that by increasing the detoxification activity of ALDH2 we would reduce A&bgr;-driven endothelial injuries and restore angiogenesis. We used a selective ALDH2 activator, Alda-1, assessing its ability to repair mitochondrial dysfunction in the endothelium. Treatment of human endothelial cells with A&bgr;1–40 (5–50 µM) induced loss of mitochondrial membrane potential, increased cytochrome c release and ROS accumulation. These events were associated with 4-HNE accumulation and decrease in ALDH2 activity (40%), and resulted in disassembly of endothelial junctions, as evidenced by &bgr;-catenin phosphorylation, disorganization of adherens and tight junctions, and by disruption of pseudocapillary formation. Alda-1 (10–40 µM) abolished A&bgr;-induced 4-HNE accumulation, apoptosis and vascular leakiness, fully restoring the pro-angiogenic endothelial phenotype and responses to FGF-2. Our data document that mitochondrial ALDH2 in the endothelium is a target for the vascular effect of A&bgr;, including loss of barrier function and angiogenesis. ALDH2 activation, by restoring mitochondrial functions in the endothelium, prevents A&bgr;-induced dysfunction and anti-angiogenic effects. Thus, agents activating ALDH2 may reduce endothelial injuries including those occurring in cerebral amyloid angiopathy, preserving the angiogenic potential of the endothelium.

Prevention of ischemic brain injury by treatment with the membrane penetrating apoptosis inhibitor, TAT-BH4.

In acute thromboembolic stroke, neurological damage is due to ischemia-induced apoptotic death of neuronal cells and the surrounding vascular network. Here, we demonstrate that the BH4 domain of the anti-apoptotic protein, Bcl-xL, attached to the membrane transport peptide, TAT, reduces stroke injury after intracerebroventricular infusion into immature rats subjected to carotid artery ligation and additional exposure to hypoxia. The injected TAT-BH4 entered neuron bodies, maintained brain architecture, protected neuronal and endothelial cells from apoptosis and promoted neuronal stem cell recruitment. In vitro, TAT-BH4 enhanced the survival of endothelial cells exposed to H2O2, increased neuronal differentiation, and induced axonal remodelling of adult neuronal stem cells. These findings indicate that TAT-BH4 administration protects against acute hypoxia/ischemia injury in the brain by preventing endothelial and neuron cell apoptosis and by inducing neuronal plasticity.

Protective Effects of Novel Metal-nonoates on the Cellular Components of the Vascular System

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.