VEGF and TGF-beta are required for the maintenance of the choroid plexus and ependyma.

Although the role of vascular endothelial growth factor (VEGF) in developmental and pathological angiogenesis is well established, its function in the adult is less clear. Similarly, although transforming growth factor (TGF) beta is involved in angiogenesis, presumably by mediating capillary (endothelial cell [EC]) stability, its involvement in quiescent vasculature is virtually uninvestigated. Given the neurological findings in patients treated with VEGF-neutralizing therapy (bevacizumab) and in patients with severe preeclampsia, which is mediated by soluble VEGF receptor 1/soluble Fms-like tyrosine kinase receptor 1 and soluble endoglin, a TGF-beta signaling inhibitor, we investigated the roles of VEGF and TGF-beta in choroid plexus (CP) integrity and function in adult mice. Receptors for VEGF and TGF-beta were detected in adult CP, as well as on ependymal cells. Inhibition of VEGF led to decreased CP vascular perfusion, which was associated with fibrin deposition. Simultaneous blockade of VEGF and TGF-beta resulted in the loss of fenestrae on CP vasculature and thickening of the otherwise attenuated capillary endothelium, as well as the disappearance of ependymal cell microvilli and the development of periventricular edema. These results provide compelling evidence that both VEGF and TGF-beta are involved in the regulation of EC stability, ependymal cell function, and periventricular permeability.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival.

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia.

Concentrations of soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng) increase in maternal blood with the approach of clinical preeclampsia. Although alterations in these circulating antiangiogenic factors herald the signs and symptoms of preeclampsia, in vitro studies suggest they may also play a role in regulating early placental cytotrophoblast functions. Early pregnancy changes in sFlt1 and sEng may thus identify women destined to develop preeclampsia. We performed a nested case-control study of 39 women who developed preeclampsia and 147 contemporaneous normotensive controls each with serum collected in the first (11 to 13 weeks of gestation) and second (17 to 20 weeks) trimesters. Whereas levels of sFlt1 and sEng at 11 to 13 weeks were similar between cases and controls (sFlt1: 3.5+/-0.3 ng/mL versus 3.0+/-0.1, P=0.14; sEng 6.9+/-0.3 ng/mL versus 6.6+/-0.2, P=0.37, respectively), at 17 to 20 weeks both were elevated in the women destined to develop preeclampsia (sFlt1: 4.1+/-0.5 ng/mL versus 3.1+/-0.1, P<0.05; sEng, 6.4+/-0.4 ng/mL versus 5.2+/-0.1, P<0.01). Women who developed preterm (<37 weeks) preeclampsia demonstrated even greater sequential changes: difference [delta{d}] between second and first trimester levels: dsFlt1, 0.63+/-0.91 ng/mL in preterm PE versus 0.05+/-0.15 in controls; dsEng, 0.73+/-0.77 ng/mL versus -1.32+/-0.18, P<0.01. Similar findings were noted in a cross-sectional analysis of specimens collected from the Calcium for Preeclampsia Prevention Study. In conclusion, sequential changes in antiangiogenic factors during early pregnancy may be useful for predicting preterm preeclampsia.

Cytomegalovirus-induced mirror syndrome associated with elevated levels of circulating antiangiogenic factors.

BACKGROUND: We describe a case where Mirror syndrome was characterized by altered levels of antiangiogenic proteins (soluble fms-like tyrosine kinase 1 [sFlt1] and soluble endoglin). CASE: We describe a pregnant patient with severe fetal and placental edema induced by congenital cytomegalovirus (CMV) infection that was associated with preeclampsia. Fetal CMV was confirmed histologically, whereas antiangiogenic factors were demonstrated to be elevated in maternal but not fetal blood. The levels of sFlt1 and soluble endoglin in our patient's serum before delivery were 116.5 ng/mL (normal pregnancy 19.3 ng/mL and preeclampsia 66.0 ng/mL, representing mean values before delivery) and 107.4 ng/mL (normal pregnancy 18.7 ng/mL and preeclampsia 52.6 ng/mL, representing mean values before delivery), respectively. In contrast, the values of sFlt1 and soluble endoglin in the cord blood were relatively low at 2.1 ng/mL and 8.2 ng/mL, respectively. CONCLUSION: If this observation is confirmed, CMV infection may be cited as a cause of Mirror syndrome and preeclampsia phenotypes associated with this disorder may be related to increased circulating antiangiogenic factors.

Lipocalin 2 antagonizes the proangiogenic action of ras in transformed cells.

Lipocalin 2 is an iron-binding secreted protein that converts embryonic kidney mesenchyme to epithelia. Previously, we reported that lipocalin 2 could revert 4T1-ras-transformed mesenchymal tumor cells to a more epithelial phenotype, increase E-cadherin expression, and suppress cell invasiveness in vitro and in vivo, indicating that lipocalin 2 is a metastasis suppressor. Here, we show that lipocalin 2 can suppress the ras-induced expression of vascular endothelial growth factor in 4T1 cells via down-regulation of ras mitogen-activated protein kinase and ras phosphatidylinositol-3-kinase signaling. In addition, the expression of thrombospondin-1 (an antiangiogenic molecule) was increased in tumors formed by 4T1-ras cells into which lipocalin 2 was stably introduced. Tumor angiogenesis, assessed via an intradermal tumor angiogenesis assay, was also suppressed by lipocalin 2. We also show that caveolin-1 is a critical mediator of this activity. These data provide new insights into the action of lipocalin 2 and raise the possibility that the administration of lipocalin 2 may be useful for inhibiting tumor angiogenesis, in addition to suppressing tumor metastasis, in cancers which show ras activation.

Soluble endoglin contributes to the pathogenesis of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-beta coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-beta1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-beta signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.

Vascular endothelial growth factor induces branching morphogenesis/tubulogenesis in renal epithelial cells in a neuropilin-dependent fashion.

Vascular endothelial growth factor (VEGF) is well characterized for its role in endothelial cell differentiation and vascular tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-121 and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-121 induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to endothelial vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.

Soluble Fms-like tyrosine kinase 1 and endothelial dysfunction in the pathogenesis of preeclampsia.

Preeclampsia, a pregnancy-specific syndrome of hypertension and proteinuria, is characterized by defective placental vasculogenesis and widespread maternal endothelial dysfunction. Although the manifestations of preeclampsia are primarily maternal, the burden of morbidity and mortality is often on the neonate, since the only effective treatment-delivery of the fetus and placenta-often results in iatrogenic prematurity. In this review, we summarize recent advances in our understanding of the pathophysiology of preeclampsia, including normal and aberrant placental vascular development and evidence for endothelial dysfunction. We describe recent evidence that supports a novel mechanism in which a maladaptive shift in placental production of angiogenic factors such as soluble fms-like tyrosine kinase 1 (a circulating antiangiogenic protein) may play an important role in the pathogenesis of preeclampsia.