Brunella Cristofaro

Identification of the prosurvival activity of nerve growth factor on cardiac myocytes

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase – PI3K – inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.

Neurotrophin p75 Receptor (p75NTR) Promotes Endothelial Cell Apoptosis and Inhibits Angiogenesis: Implications for Diabetes-Induced Impaired Neovascularization in Ischemic Limb Muscles

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75NTR), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75NTR expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75NTR gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75NTR depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin], BIRC5 (survivin), PTTG1 (securin) and VEZF1. Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75NTR. Collectively, our data newly demonstrate the antiangiogenic action of p75NTR and open new avenues for the therapeutic use of p75NTR inhibition to combat diabetes-induced microvascular liabilities.

Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization.

Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease.

Endothelial Nuclear Factor-κB–Dependent Regulation of Arteriogenesis and Branching

Background— Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. Methods and Results— We used an inhibitor of NF&kgr;B activation (I&kgr;B&agr;SR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-&kgr;B activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-&kgr;B activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1&agr; levels, and a marked decrease in &dgr;-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. Conclusions— These findings identify nuclear factor-&kgr;B as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-&kgr;B achieves this by regulating hypoxia-inducible factor-1&agr;–dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating &dgr;-like ligand 4 expression, which in turn determines the size and complexity of the network.

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4+/- mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4+/- mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Neurotrophin-3 Is a Novel Angiogenic Factor Capable of Therapeutic Neovascularization in a Mouse Model of Limb Ischemia

Objective—To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth. Methods and Results—We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3–induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase–Akt kinase–endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3. Conclusion—Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease.

Type-2 diabetic Lepr(db/db) mice show a defective microvascular phenotype under basal conditions and an impaired response to angiogenesis gene therapy in the setting of limb ischemia.

Diabetes mellitus is associated with macro- and micro-angiopathy, leading to increased risk of peripheral ischemia. In the present study, we have characterized the microvascular phenotype at the level of limb muscles and the spontaneous angiogenesis response to surgically-induced unilateral limb ischemia in a murine model of type-2 diabetes, the obese C57BL/KsOlaHsd-Lepr(db/db) mice (Lepr(db/db)), and in non-diabetic heterozygous Lepr(db/+). Wild type C57BL mice (WT) were used as controls. The basal microvascular phenotype was determined in mice aged 3 or 5 months, while the response to limb ischemia was studied only in 5-month old mice. Moreover, in 5-month old ischemic Lepr(db/db) and Lepr(db/+), we have tested the therapeutic potential of local angiogenesis gene therapy with human tissue kallikrein (hTK) or constitutively-activated Akt kinase (Myr-Akt). We found that in the muscles of 3- or 5-month old Lepr(db/db), apoptosis of endothelial cells was enhanced and the densities of capillary and arteriole were reduced. Arterioles of Lepr(db/db) showed hypertrophic remodelling and, occasionally, lumen occlusion. Following ischemia, Lepr(db/db) showed a defective reparative angiogenesis in ischemic muscle, delayed blood flow recovery, and worsened clinical outcome as compared with controls. Five-month old Lepr(db/+) displayed an increase in endothelial cell apoptosis under basal conditions, while capillary and arteriole densities were normal. Lepr(db/+) mounted a proper reparative angiogenesis response to limb ischemia and regained blood flow to the ischemic limb, regularly. Local gene therapy with hTK or Myr-Akt induced angiogenesis in ischemic muscles of Lepr(db/+) and Lepr(db/db). However, in the Lepr(db/db) neither gene therapy approach improved the blood flow recovery and the clinical outcome from ischemia. In contrast, either hTK or Myr-Akt gene transfer improved the post-ischemic recovery of Lepr(db/+). Type-2 diabetes has a negative impact on the basal microvascular phenotype and severely impairs post-ischemic recovery of limb muscles. Gene therapy-induced stimulation of neovascularization might not suffice as a sole therapeutic strategy to combat type-2 diabetes-related vascular complications. In type-2 diabetic patients, therapeutic angiogenesis may need to be further optimized before being recommended for clinical applications.

Netrin-1 controls sympathetic arterial innervation

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Possible novel targets for therapeutic angiogenesis.

An increasing number of studies about the molecular basis of angiogenesis are rapidly disclosing novel signal pathways involved in the blood vessel formation process. This review will focus on bone morphogenic proteins, Hedgehog, Notch, ephrins, neuropilins, neurotrophins and netrins. These recently discovered angiogenesis mediators are involved in vascular development during embryogenesis and, interestingly, they are shared between the nervous and vascular systems. They represent new potential targets in the vasculature and suggest novel therapeutic opportunities.

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4+/− mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4+/− CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.