VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling

Objective— Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results— sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. Conclusions— These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Infection with Anaplasma phagocytophilum activates the phosphatidylinositol 3-Kinase/Akt and NF-κB survival pathways in neutrophil granulocytes

Anaplasma phagocytophilum, a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-?B signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum-induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-?B in A. phagocytophilum-infected neutrophils. Activation of NF-?B leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum-infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum-infected neutrophils.

Dysregulation of placental cystathionine-β-synthase promotes fetal growth restriction

Fetal growth restriction (FGR) is characterized by the birth weight and body mass below the tenth percentile for gestational age. FGR is a major cause of perinatal morbidity and mortality and babies born with FGR are prone to develop cardiovascular diseases later in life. The underlying pathology of FGR is inadequate placental transfer of nutrients from mother to fetus, which can be caused by placental insufficiency. Hydrogen sulfide (H2S), a gaseous messenger is produced endogenously by cystathionine-lyase (Cth), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), which are present in human placenta. Recently, we demonstrated that the dysregulation of H2S/Cth pathway is associated with preeclampsia and blockade of CSE activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in H2S pathways promote FGR and H2S donor restores fetal growth in mice where CBS or CSE activity has been compromised. Western blotting and qPCR revealed that placental CBS expressions were significantly reduced in women with FGR. ELISA analysis showed reduced placental growth factor production (PlGF) from first trimester (8–12 weeks gestation) human placental explants following inhibition of CBS activity by aminooxyacetic acid (AOA). Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction. This was associated with reduced PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor treated mice. These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia.